Chrysosporium Cellulase and Methods of Use

ABSTRACT

The subject invention relates to novel compositions of neutral and/or alkaline cellulase and methods for obtaining neutral and/or alkaline cellulase compositions from  Chrysosporium  cultures, in particular  Chrysosporium lucknowense.  This invention also provides mutants and methods of generating mutants of  Chrysosporium  capable of producing neutral and/or alkaline cellulase. This invention also relates to the genes encoding the enzymes comprising the neutral and/or alkaline cellulase composition. In addition, this invention provides methods of culturing  Chrysosporium  to produce neutral and/or alkaline cellulases. The neutral and/or alkaline cellulase compositions of the subject invention can be used in a variety of processes including stone washing of clothing, detergent processes, deinking, color brightening, depilling and biobleaching of paper and pulp and treatment of waste streams. The present invention also relates to the isolation and purification of cellulase enzymes, having glucanase and cellobiohydrolase activity, and useful for stonewashing applications.

FIELD OF THE INVENTION

This invention relates to neutral and/or alkaline cellulases and novelmethods for producing the same. More specifically this invention relatesto cellulases produced by fungi of the genus Chrysosporium, andparticular strains of Chrysosporium lucknowense. This invention alsorelates to industrial uses for these neutral or alkaline cellulases andcompositions comprising the same.

BACKGROUND OF THE INVENTION

Clothing made from cellulosic fabrics such as cotton, linen, hemp,ramie, cupro, lyocell, newcell, rayon, polynosics, are very popular. Ofparticular interest are clothing items such as jeans made fromindigo-dyed denim fabrics made of cotton or cotton blends. Such clothingitems are typically sewn from sized and cut cloth and tend to be stiffdue to the presence of sizing compositions. In other cases the fibers orrolls of fabric are treated with enzymes prior to sewing the finalgarment. After a period of wear, the clothing items can develop acertain degree of softness, an overall reduction of shade as well aslocalized areas of color variation. Additionally, after repeated washingthe garment continues to provide a more comfortable fit, a softer feeland a worn appearance. In recent years such comfort, feel and appearancehave become increasingly popular.

The most widespread methods for producing this comfort, feel and lookinvolve washing of clothing items with cellulases in large washingmachines with pumice stones or other abrasives. The pumice helps softenthe fabric and helps to provide the faded surface similar to thatproduced by the extended wear of the fabric. However, the use of pumicehas some disadvantages. For example, the pumice must be manually removedfrom processed clothing items because it tends to accumulate in pockets,on interior surfaces, in creases, and in folds. Also, the pumice stonescan cause overload damage to electric motors of stone washing machines,and clog machine drainage passages and drain lines. These processing andequipment problems can add significantly to the cost of doing businessand to the purchase price of the goods.

In view of the problems of using pumice, alternative methods to usingpumice or other abrasives in the stone washing process have been sought.One alternative involves the use of enzyme treatments which break downthe cellulose in fabrics (Geller U.S. Pat. No. 4,951,366; Olson U.S.Pat. Nos. 4,832,864, 4,912,056, Olson et al. U.S. Pat. Nos. 5,006,126,5,122,159 and 5,213,581, Christner et al. U.S. Pat. No. 4,943,530, Boeghet al. U.S. Pat. No. 4,788,682). Methods for treating cellulosecontaining fabrics with hydrolytic enzymes, such as cellulases, areknown in the art to improve the softness or feel of such fabrics (NovoBrochure Cellulase SP 227; Novo Brochure Celluzyme; Murata U.S. Pat. No.4,443,355; Parslow U.S. Pat. No. 4,661,289; Tai U.S. Pat. No. 4,479,881;Barbesgaard U.S. Pat. No. 4,435,307; Browning UK. Patent No. 1,368,599).

Cellulases are known in the art as enzyme systems that hydrolyzecellulose (β-1,4-glucan linkages), thereby resulting in the formation ofglucose, cellobiose, cellooligosaccharides, and the like. Cellulasecompositions are comprised of several different enzyme components,including those identified as exocellobiohydrolases, endoglucanases, andβ-glucosidases. Moreover, these classes of enzymes can be furtherseparated into individual isoenzymes.

The complete cellulase system is required to efficiently convertcrystalline cellulose to glucose. Generally, if total hydrolysis of acellulose substrate is needed, the cellulase mixture should containβ-glucosidases and cellobiohydrolases, as well as endoglucanases.Endoglucanases catalyze random hydrolysis of β-1,4-glycosidic bondsbetween glucose units of cellulose polymers. Such components hydrolyzesoluble cellulose derivatives such as carboxymethylcellulose, therebyreducing the viscosity of such solutions. Such enzyme components act oninternal regions of the polymer, resulting in a rapid decrease inaverage polymer chain length together with a slow increase in the numberof reducing ends. The rapid decrease in average chain length of thecellulose polymer is evidenced by the decrease in viscosity of acellulose solution.

The substrate specificity and mode of action of the different cellulasesvaries among strains of organisms that produce cellulases. For example,the currently accepted mechanism of cellulase action in cellulase fromthe fungus Trichoderma reesei is that endoglucanase activity first-breakinternal β-1,4-glucosidic bonds in regions of low crystallinity of thecellulose (Ruohnen L., et al. In: “Proceedings of the Second TricelSymposium on Trichoderma Reesei Cellulases and Other Hydrolases”, (ed.by P. Sudminen and T. Reinkainen.,) Foundation for Biotechnology andIndustrial Fermentation Research 8; (1993):87-96) The cellobiohydrolaseactivity binds preferentially to the crystalline regions of thenon-reducing end of the cellulose to release cellobiose as the primaryproduct. β-Glucosidase or cellobiase activities then act oncellooligosaccharides, e.g., cellobiose, to give glucose as the soleproduct.

Cellulases are produced in fungi, bacteria, and other microbes. Fungitypically produce a complete cellulase system capable of degradingcrystalline forms of cellulose. For example, Trichoderma reesei producesand secreates all of the enzyme activities needed for efficientbreakdown of crystalline cellulose, namely endo-1,4-β-D-glucanases,cellobiohydrolases (exo-1,4-β-D-glucanases), and 1,4-β-D-glucanases, orβ-glucosidases. Fungal cellulases have an added advantage in thatcellulases in fungi can readily be produced in large quantities viafermentation procedures.

Cellulases, or the components thereof, are known in the art to be usefulin a variety of industrial textile applications in addition to the stonewashing process. For example, cellulases are used in detergentcompositions, for the purpose of enhancing the cleaning ability of thecomposition, as a softening agent, for color brightening, depilling andother uses. When so used, the cellulase will degrade a portion of thecellulosic material, e.g., cotton fabric, in the wash, which facilitatesthe cleaning and/or softening of the fabric. The endoglucanasecomponents of fungal cellulases have also been used for the purposes ofenhancing the cleaning ability of detergent compositions, for use as asoftening agent, and for use in improving the feel of cotton fabrics,and the like. However, there is a problem with using the cellulasederived from Trichoderma spp. and especially Trichoderma longibrachiatumin detergent compositions. Generally, such components have their highestactivity at acid pHs whereas most laundry detergent compositions areformulated for use at neutral or alkaline conditions.

Other textile applications in which cellulases have been used includesoftening (Browning, UK Patent No. 1,368,599, Parslow, U.S. Pat. No.4,661,289, Tai U.S. Pat. No. 4,479,881 and Barbesgaard, U.S. Pat. No.4,435,307), defibrillation (Gintis, D. Mead, E. J., Textile ResearchJournal, 29, 1959; Cooke, W. D., Journal Of The Textile ResearchInstitute, 74, 3, 1983; Boegh, European Patent Application No. 0 220016). Cellulases have also been used in combination with a polymericagent in a process for providing localized variation in the colordensity of fibers. (WO/94/19528 and WP/94/1529).

Cellulases are classified in the garment and textile industry accordingto their pH range of operation. Acid cellulases typically have theirpeak activity at pH values of about 4.0 to 5.5 and less, neutralcellulases at about pH 5.5 to 7.5, and alkaline cellulases at about pH7.5 to 11.0. Some enzyme compositions may have broader ranges ofoperation. For example, the neutral/alkaline cellulases may operate atacid, neutral and alkaline pH's at between about 40° C. to 60° C.

Acid, neutral and alkaline cellulases are typically used in the “stonewash” treatment of denim jeans, with or without surfactants, buffers,detergents, anti-redeposition agents, softening agents, pumice stones orother abrasives, bleaching agents, such as optical bleaching agents,enzymes, or other means. If the cellulase composition is not formulatedand/or pre-buffered then for acid cellulases, the pH is typicallyadjusted to between pH 4.5-5.5, with for example, a sodium citrate andcitric acid buffer, and for neutral or alkaline cellulases between5.5-7.5 with, for example, a monosodium and disodium phosphate buffer.Neutral and alkaline cellulases are typically used as additives tolaundry detergents where the pH of operation may range from about pH 7.0to 11.5. In stone wash applications typical acid cellulases generallyprovide greater backstaining or redeposition of the indigo dye andgreater strength loss of the fabric while the typical neutral andalkaline cellulases generally provide less abrasion, lower backstainingor redeposition and less strength loss of the fabric.

The neutral/alkaline cellulases are the most preferred type ofcellulases for the stonewash industry because they cause lower levels ofbackstaining or redeposition and lower strength loss than acidcellulases (ie; from Trichoderma sp.). Furthermore, neutral/alkalinecellulases, unlike their acid counterparts, operate at a much wider pHrange and are able to maintain better relative wash performance within awider pH range (pH 5.0-pH 8.0) in the stone washing industry. Therefore,neutral/alkaline cellulases provide several advantages. First, theincoming feed water in wet processing facilities is typically withinthis pH range lessening the need for as precise pH control as comparedto acid cellulases. This makes the stonewashing process more tolerant tooperator pH error or neglect leaving the overall procedure moreforgiving than procedures using acid cellulases. Secondly, it is knownthat denim fabrics are alkaline in nature owing to the fact that thedyeing process utilities caustic soda. Simply washing denim releasesthis caustic into the wash water and the pH of the wash water generallyrises. The alkalinity may overcome the bath buffers, but the effect ofincreased pH is less severe on neutral/alkaline cellulases compared toacid cellulases because neutral/alkaline cellulases operate not only athigher pH, but also over a wider pH range.

The wide spectrum of industrial uses for cellulases or the components ofcellulases, especially alkaline and/or neutral cellulases, establishes aclear need for cellulases that are operative at neutral and/or alkalinepH. The present invention provides a procedure for producingneutral/alkaline cellulases having enzymatic activity at neutral and/oralkaline pH's and compositions comprising the same.

SUMMARY OF THE INVENTION

This invention relates, in general, to neutral and/or alkalinecellulases and novel methods for producing the same. More specifically,the subject invention provides a method for producing cellulasecompositions from fungi of the genus Chrysosporium, and particularChrysosporium lucknowense, wherein the cellulase compositions haveenzymatic activity at neutral and/or alkaline pH's. Industrialapplications for the cellulase composition are also provided.

One embodiment of this invention relates to isolated and purifiedcultures of wild type and mutant fungi of the genus Chrysosporiumcapable of producing neutral and/or alkaline cellulase compositions, inparticular to the strain Chrysosporium lucknowense—GARG 27K and mutantsthereof.

Yet another embodiment of this invention provides culturing conditionsfor producing neutral or alkaline cellulases from fungi of the genusChrysosporium.

In a further embodiment, this invention provides methods to producing aneutral and/or alkaline cellulase composition through recombinanttechnology from fungi of the genus Chrysosporium.

In yet a further embodiment of this invention methods for generating andculturing mutant strains of the fungi Chrysosporium capable of producingneutral and/or alkaline cellulase are provided.

Another embodiment of this invention relates to the nucleic acidsequences encoding the enzymes of the cellulases compositions producedby Chrysosporium or genetically modified strains of Chrysosporium.

Another embodiment relates to the purified and isolated enzymes of thecellulase compositions produced by Chrysosporium or genetically modifiedstrains of Chrysosporium.

In yet another embodiment of this invention methods of use are providedfor alkaline and/or neutral cellulases produced by Chrysosporium intextile applications, such as softening, bleaching and stone washingprocedures, garment dyeing applications, defibrillation, orbiopolishing, color brightening and depilling.

Another embodiment of this invention relates to detergent compositionscomprising Chrysosporium cellulase in detergent preparations.

Another embodiment of this invention is to provide methods of use forthe cellulase compositions in the saccharification of lignocellulosebiomass from agriculture, forest products, municipal solid waste, andother sources.

Yet other embodiments of this invention involve the use of the cellulasecompositions for production of fuels and other chemicals for thebiobleaching of wood pulp, and for de-inking of recycled print paper.

DETAILED DESCRIPTION OF THE INVENTION

As utilized herein, reference to a “neutral-alkaline cellulase” refersto a cellulase composition which retains significant enzymatic activityat pH values of about 5.5 and above. In a preferred embodiment, theneutral and/or alkaline cellulase compositions of the subject inventionhave peak enzymatic activity between about pH 5.5 to about 7.5 at 40° C.to about 60° C. In the event that the peak enzymatic activity is at a pHof less than about 5.5, the neutral-alkaline cellulase composition willhave at least about 50% of the optimal enzymatic activity at about pH6.0 to about 7.0 at about 40° C. to about 60° C. By way of example suchactivities may be measured by RBBCMCase, CMCase, Cellazyme,endoviscometric or filter paper activity (FPA). Thus, the cellulasecompositions of the subject invention will have useful enzymaticactivity at pHs greater than 5.5 such that the enzyme composition can beused in stone wash, detergent, de-inking or other applications whereneutral and/or alkaline cellulase activity is needed.

The subject invention relates to compositions of cellulases having highactivity at neutral or alkaline pH's and to unique methods for producingsaid neutral and alkaline cellulase compositions. The neutral/alkalinecellulase compositions of this invention may be obtained from anyspecies of Chrysosporium. In a particularly preferred embodiment, thecellulase compositions of the present invention are isolated fromChrysosporium lucknowense Garg 27K (designated isolate C1) depositedunder the Budapest Treaty with the International Depository at theAll-Russian Collection of Microorganisms of the Russian Academy ofSciences, Bakhrushina St. 8: Moscow, Russia 113184, on Aug. 29, 1996,and assigned accession number VKM F-3500D. The cellulase compositions ofthe subject invention are highly advantageous because they possessenzymatic activity at neutral and/or alkaline pH thereby providingbeneficial performance characteristics in industrial applications.

The cellulase compositions prepared from fungal strains of the subjectinvention exhibit activity at between about pH 5.0 to about 12.0 atbetween about 40° to 60° C. as determined by a CMCase, RBBCMCase,Cellazyme, endoviscometric or Filter Paper Activity (FPA) assays. In apreferred embodiment for a stone wash procedure, the cellulasecomposition may have optimal activity at between about pH 5.5 to 7.0 atabout 40° C. to about 60° C. Good performance activity at neutral andalkaline pH (ie: 6.0, 7.0 & 8.0) has been demonstrated for the neutraland/or alkaline cellulases of the instant invention in Stonewashapplication trials and at pH 10.0 and above for detergent applicationtrials.

The fermentation procedures for culturing cellulolytic microorganismsfor production of cellulase are known in the art. For example, cellulasesystems can be produced either by solid or submerged culture, includingsolid state, batch, fed-batch, and continuous-flow processes. Thecollection and purification of the cellulase systems from thefermentation broth can also be effected by procedures known in the art.The cellulase composition is readily isolated from the fungal cultureby, for example, centrifugation or filtration steps and concentration ofthe filtrate via membrane or hollow fibers ultrafiltration equipment.

The fungal strain Chrysosporium used to produce the cellulasecompositions of the subject invention can be cultured according tostandard methods and conditions known in the art. In a preferredembodiment, the cellulase composition of the subject invention isobtained from the C1 strain. The C1 Chrysosporium strain may be grown ina medium containing inorganic salts, organic nitrogen sources, such aspeptones, defatted cotton seed flour, corn steep liquor, or yeastextract and carbon source. Examples of carbon source include, but is notlimited to, glucose, lactose, sucrose, cellulose or other carbohydrates.More preferably, the fungal strain is grown in media containing bothlactose and peptone or lactose and yeast extract. By way of example thefermentation media can compose lactose at about 0.3% to about 1.0%,preferably about 0.5% to about 0.6%, peptone at about 0.3% to about1.0%, preferably about 0.5% to about 0.6%. Other nitrogen sources andcarbohydrate sources known in the art may be used in the fungal growthmedia including, but not limited to, sweet beet pulp, barley malt, wheatbran, and others known in the art. By way of example sweet beet pulpconcentrate may be used in a range of about 15 to about 30 grams/liter(g/L), preferably about 20 to about 25 g/L; barley malt may be used in arange about 10 g/L to about 20 g/L, preferably about 14 g/L or about 16g/L, wheat bean may be used in a range about 3 g/L to about 8 g/L,preferably about 5 g/L to about 6 g/L. In one embodiment, the C1 strainis cultured in rotated shake flasks in saline medium containing sweetbeet pulp, barley malt, and wheat bran. Cellulase compositions may beisolated from fungi cultured about 3 to 7 days in a growth medium bycentrifugation and ultrafiltration concentration of the cell culturemedium.

Alternatively the Chrysosporium cultures can be cultured on a largescale for commercial use, by using conventional fermentation techniques.In this context fermentation is used broadly to refer to any controlledfungal culturing conditions. Prior to large scale growth an inoculum ofsaid growth culture is generally cultured. The inoculum media maycontain conventional ingredients including, but not limited to, carbonsources, organic nitrogen sources, and inorganic salts. Carbon sourcesmay include, but are not limited to, glucose, lactose, glycerol, and/orcellulose at concentrations in the range of about 0.5 to 200 g/L, morepreferably in the range of about 5 to 50 g/L. Organic nitrogen sourcesmay include, but are not limited to, yeast extract, peptone, or defattedcotton seed flour at concentrations in the range of about 0.5 to 30 g/L,more preferably in the range of 5 to 15 g/L. Inorganic salts mayinclude, but are not limited to, potassium phosphate, for example atabout 0.01 to about 10 g/L, magnesium sulfate, for example at about 0.01to 3.0 g/L, ferrous sulfate, for example at about 0.001 to 10 mg/L.

An inoculum or starter culture may be used to initiate the Chrysosporiumculture for a fermenter by methods known in the art. The media used forfermentation may comprise conventional ingredients for culturing fungi,including but not limited to, cellulose, organic nitrogen sources,magnesium chloride and calcium chloride. Examples of organic nitrogensources include, but are not limited to, peptone or defatted cotton seedflour, such as Pharmamedia.

By way of example, the media may comprise about 5 g/l to about 20 g/L ofpeptone or defatted cotton seed flour, about 10 g/L to about 30 g/L ofcellulose, about 0.03 g/L to about 0.06 g/L of magnesium sulfateheptahydrate and about 0.4 g/L to about 0.8 g/L of calcium chloridedihyrate.

One of skill in the art will appreciate that during fermentation thetemperature, oxygenation, pH, and nutrient levels of fermentationmixture should be maintained. By way of example, dissolved oxygen levelsshould be maintained at about 10 to 60% of air saturation, preferably atabout 20 to 40% of air saturation. The pH should be maintained betweenabout 5 and 8, preferably between about 6.5 and 7.5, most preferablybetween 6.9 and 7.1 and the temperature may be maintained at betweenabout 25° C. to about 40° C., preferably at about 28° C. to 35° C. Thefeed solution may comprise ingredients similar to the fermentation mediabut at higher concentrations to minimize dilution when added to thefermentation media.

The cellulase compositions produced according to the methods of thesubject invention are useful for a variety of other applications forwhich cellulase activity, in particular neutral and/or alkalinecellulase activity, is needed. In one embodiment of this invention, theneutral and/or alkaline cellulase compositions can be used in stonewashing procedures for denim jeans. By way of example, the mostpreferred pH range of stone wash applications is between about 5.5 to7.5, most preferably at about pH 6 to about 7. The neutral and/oralkaline cellulase composition obtained from Chrysosporium isolatesadvantageously have significant enzymatic activity at or above neutralor alkaline pH. Stone wash procedures conducted with neutral and/oralkaline cellulase run at neutral and/or alkaline pH's are particularlyadvantageous compared to traditional procedures using acid cellulases(eg: those from Trichoderma reesei) because of lower levels ofbackstaining on the garments, less strength loss to the garments and thealkalinity of the water that is present naturally during this process.These stone washing procedures result in jeans with highly desirablefeel and appearance. By way of example, 0.02 to 10 g of cellulasepreparation 47.0528 described herein, may be used per 135 g of denim.One of skill in the art will know how to regulate the amount orconcentration of the cellulase composition produced by this inventionbased on such factors as the activity of the cellulase, and the washconditions, including but not limited to temperature and pH.

In yet another embodiment of this invention, the cellulase compositionsof this invention can be used to reduce or eliminate the harshnessassociated with fabrics made from cellulose by addition to detergentcompositions. By way of example, the preferred range for detergentcompositions is between about pH 8 to about 12, most preferably pH 10 toabout 11. The cellulase compositions of the subject invention can beused in detergent compositions at neutral and or alkaline pH. Detergentingredients contemplated for use with the cellulase composition of thesubject invention include any detergent ingredient known in the art.Examples of such ingredients include, but are not limited to,detergents, buffers, surfactants, bleaching agents, softeners, solvents,solid forming agents, abrasives, alkalis, inorganic electrolytes,cellulase activators, antioxidants, builders, silicates, preservatives,and stabilizers, and are known in the art. The detergent compositions ofthis invention preferably employ a surface active agent, i.e.,surfactant, including anionic, non-ionic, and ampholytic surfactantswell known for their use in detergent compositions. In addition to thecellulase components and the surface active agent, the detergentcompositions of this invention can additionally contain one or more ofthe following components; the enzymes amylases, cellulases, proteinase,lipases, oxido-reductases, peroxidases and other enzymes; cationicsurfactants and long-chain fatty acids; builders; antiredepositionagents; bleaching agents; bluing agents and fluorescent dyes; cakinginhibitors; masking agents for factors inhibiting the cellulaseactivity; cellulase activators; antioxidants; and solubilizers. Inaddition, perfumes, preservatives, dyes, and the like can be used, ifdesired, with the detergent compositions of this invention. Examples ofdetergent compositions employing cellulases are exemplified in U.S. Pat.Nos. 4,435,307; 4,443,355; 4,661,289; 4,479,881; 5,120,463, which areherein incorporated by reference.

When a detergent base used in the present invention is in the form of apowder, it may be one which is prepared by any known preparation methodincluding a spray-drying method and/or a granulation method. Thegranulation method are the most preferred because of the non-dustingnature of granules compared to spray dry products. The detergent baseobtained by the spray-drying method is hollow granules which areobtained by spraying an aqueous slurry of heat-resistant ingredients,such as surface active agents and builders, into a hot space. Thegranules have a size of from about 50 to about 2000 micrometers. Afterthe spray-drying, perfumes, enzymes, bleaching agents, and/or inorganicalkaline builders may be added. With a highly dense, granular detergentbase obtained by such as the spray-drying-granulation method, variousingredients may also be added after the preparation of the base. Whenthe detergent base is a liquid, it may be either a homogenous solutionor an inhomogeneous solution.

The cellulase compositions of this invention preferably exhibit highlevels of activity at alkaline or neutral pH's, but also may exhibitenzymatic activity at acidic pH's. Therefore, the detergent compositionscomprising the cellulases of the present invention can be used in abroad pH range of from acidic to alkaline pH.

Other textile applications in which these cellulase compositions may beused include, but are not limited to, Garment Dyeing applicationsincluding but not limited to Enzymatic Mercerizing of viscose,Bio-Polishing applications, Enzymatic Surface Polishing; Biowash(washing or washing down treatment of textile materials), EnzymaticMicrofibrillation, Enzymatic “cottonization” of linen, ramie and hemp;and treatment of Lyocel or Newcell (ie; “TENCEL” from Courtauld's),Cupro and other cellulosic fibers or garments, dye removal from dyedcellulosic substrates such as dyed cotton (Leisola & Linko—(1976)Analytical Biochemistry, v. 70, p. 592. Determination Of TheSolubilizing Activity Of A Cellulose Complex With Dyed Substrates; Blum& Stahl—Enzymic Degradation Of Cellulose Fibers; Reports of the ShizuokaPrefectural Hamamatsu Textile Industrial Research Institute No. 24(1985)), as a bleaching agent to make new indigo dyed denim look old(Fujikawa—Japanese Patent Application Kokai No. 50-132269), to enhancethe bleaching action of bleaching agents (Suzuki—Great Britain PatentNo. 2 094 826), and in a process for compositions for enzymatic desizingand bleaching of textiles (Windbichtler et al., U.S. Pat. No. 2,974,001.Another example of enzymatic desizing using cellulases is provided inBhatawadekar (May 1983) Journal of the Textile Association, pages 83-86.

In other industrial embodiments, the cellulase compositions can be usedin the saccharification of lignocellulose biomass from agriculture,forest products, municipal solid waste, and other sources, for theproduction of fuels and other chemicals through fermentation, forbiobleaching of wood pulp, and for de-inking of recycled print paper allby methods known to one skilled in the art.

In yet another embodiment of the subject invention, various componentsof the neutral and alkaline cellulase compositions can be isolated andused independently of each other. Specific components or cellulasecomposition enriched by certain cellulase components can be produced orisolated by chemical and physical means from mutants or specificallyproduced by genetic engineering methods. The cellulase system can bepurified into separate components by art-recognized separationtechniques including ion exchange chromatography at a suitable pH,affinity chromatography, size exclusion, chromatography and like. Forexample, in ion exchange chromatography, it is possible to separate thecellulase components by eluting with a pH gradient, or a salt gradient,or both. Such separations can be done by those skilled in the art havingthe benefit of the teachings provided herein.

Once the individual enzymatic components of the cellulase compositionare fractionalized and isolated the proteins may be partially sequencedor microsequenced to design synthetic DNA or probes to isolate the geneencoding the enzymatic proteins of interest. Generally the aminoterminal sequence of the protein is determined by conventional proteinsequencing methods or by automated sequence (Ausubel et al., (1987) in“Current Protocols in Molecular Biology”, John Wiley and Sons, New York,N.Y.). Alternatively, other regions of the protein may be sequenced incombination with chemical cleavage or enzymatic cleavage and proteinseparation techniques. (Ausubel et al., (1987) in “Current Protocols inMolecular Biology”, John Wiley and Sons. New York, N.Y.). One of skillin the art will understand that the synthetic DNA clones or probes canbe used in routine cloning techniques to isolate the genes correspondingto the enzymes present in the neutral/alkaline cellulase compositionsproduced by Chrysosporium.

It will be understood by one skilled in the art that nucleic acidsequences obtained by this invention in the art may vary due to thedegeneracy of the genetic code variations in the DNA sequence, but willstill result in a DNA sequence capable of encoding the enzymaticcomponents of the cellulase compositions. Such DNA sequences aretherefore functionally equivalent to the nucleic acid sequences of theinstant invention and are intended to be encompassed within the presentinvention. Also intended to be encompassed within this invention arenucleic acid sequences which are complementary to nucleic acid sequencescapable of hybridizing to the disclosed nucleic acid sequence under avariety of conditions.

This invention further includes the nucleic acid sequences encoding theenzymes of the cellulase compositions of this invention and thoseproteins or peptides having substantially the same function as theenzymatic proteins or peptides of this invention. Such proteins orpolypeptides include, but are not limited to, a fragment of the protein,or a substitution, addition or deletion mutant. This invention alsoencompasses proteins or peptides that are substantially homologous tothe proteins encoding the enzymes comprising the cellulase compositionof this invention. The term “analog” includes any polypeptide having anamino acid residue sequence substantially identical to the sequencespecifically in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays thefunctional aspects of the proteins as described herein. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, nu leucine or alaninefor another, the substitution of one polar (hydrophilic) residue foranother such as between arginine and lysine, between glutamine andasparagine, between threonine and serine, the substitution of one basicresidue such as lysine, arginine or histidine for another, or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

Proteins or polypeptides of the present invention also include anypolypeptide having one or more additions and/or deletions or residuesrelative to the sequence of a polypeptide whose sequence is included inthe proteins of this invention so long as the requisite activity ismaintained.

This invention also provides a recombinant DNA molecule comprising allor part of the nucleic acid sequences isolated by this invention and avector. Expression vectors suitable for use in the present inventioncomprise at least one expression control element operationally linked tothe nucleic acid sequence. The expression control elements are insertedin the vector to control and regulate the expression of the nucleic acidsequence. Examples of expression control elements include, but are notlimited to, lac system, operator and promoter regions of phage lambda,yeast or other fungi promoters. Examples of promoters that may be usedinclude, but are not limited to, glucoamylase. Additional preferred orrequired operational elements include, but are not limited to, leadersequence, termination codons, polyadenylation signals and any othersequences necessary or preferred for the appropriate transcription andsubsequent translation of the nucleic acid sequence in the host system.It will be understood by one skilled in the art the correct combinationof required or preferred expression control elements will depend on thehost system chosen. It will further be understood that the expressionvector should contain additional elements necessary for the transfer andsubsequent replication of the expression vector containing the nucleicacid sequence in the host system. Examples of such elements include, butare not limited to, origins of replication and selectable markers. Itwill further be understood by one skilled in the art that such vectorsare constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology”, John Wiley and Sons, New York,N.Y.) or may be commercially available.

Another aspect of this invention relates to a host organism into whichrecombinant expression vector containing all or part of the nucleic acidsequence has been inserted. The host cells transformed with the nucleicacid sequence of this invention includes eukaryotes, such as animal,plants or seeds, insect and yeast cells, fungal cells, and prokaryotes,such as E. coli or other bacteria. Examples of fungal host cells includebut are not limited to Aspergillus, Trichoderma, Humicola, Periicillium,or Neurospora. The means by which the vector carrying the gene may beintroduced into the cell include, but are not limited to,transformation, microinjection, electroporation, transduction, ortransfection using DEAE-dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art (Sambrook et al. (1989) in“Molecular Cloning. A Laboratory Manual”, Cold Spring Harbor Press,Plainview, N.Y.). Alternatively, Chrysosporium cells can be transformedwith the nucleic acid sequence of this invention to amplify productionof cellulases by Chrysosporium.

In a preferred embodiment, expression vectors that function in fungalcells are ased. Examples of such vectors include, but are not limited toplasmids, described in the patents (Ogawa; Japanese patent JP5095787 A930420, Ozeki; Japanese patent JP7115976 A 950509, Murakami; Japanesepatent JP3094690 A 910419, Ishida; Japanese patent JP3251175 A 911108,Uozumi; Japanese patent JP5268953 A 931019 DW9346 C12N-009/34 011pp,Gottschalk; German patent DE3908813 A 900920 DW9039 000 pp, Gysler;European patent EP-683228 A2 951122 DW9551 C12n-015/60 Eng 041 pp). Itis preferred that the recombinant protein expression vector isintroduced into fungal cells, such to ensure proper processing andmodification and modification of the introduced protein.

In a further embodiment, the recombinant protein expressed by the hostcells can be obtained as a crude lysate or can be purified by standardprotein purification procedures known in the art which may includedifferential precipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. (Ausubel et. al., (1987) in“Current Protocols in Molecular Biology” John Wiley and Sons, New York,N.Y.). In the case of immunoaffinity chromatography, the recombinantprotein may be purified by passage through a column containing a resinwhich has bound thereto antibodies specific for the protein of interest(Ausubel et. al., (1987) in “Current Protocols in Molecular Biology”John Wiley and Sons, New York, N.Y.).

All or parts of the nucleic acid sequences of this invention can also beused as probes to isolate other homologs in other genera or strains. Ina preferred embodiment the nucleic acid sequences are used to screen aChysosporium library; positive clones are selected and sequenced.Examples of sources from which the gene library can be synthesizedinclude, but are not limited to species of Chlysosporium, Aspergillus,Penicillium, Humicola, Cephalosporium Tricoderma or bacteria such asBacillus. One skilled in the art will understand the appropriatehybridization conditions to be used to detect the homologs. Conventionalmethods for nucleic acid hybridization, construction of libraries andcloning techniques are described in Sambrook et al., (eds) (1989) In“Molecular Cloning A Laboratory Manual” Cold Spring Harbor Press,Plainview, N.Y. and Ausubel et al., (eds) in “Current Protocols inMolecular Biology” (1987), John Wiley and Sons, New York, N.Y.

This invention also relates to mutant strains of Chrysosporium, inparticular, mutant strains of Chrysosporium lucknowense capable ofproducing neutral and/or alkaline cellulases. Methods of DNA mutagenesisand screening for mutants are well known to these skilled in the art andinclude a variety of chemical and enzymatic methods. Examples of suchmethods include but are not limited to exposure of the fungi toultraviolet light (UV), nitrous acid,N-methyl-N′-nitro-N-nitrosoguanidine (NG), and 4-nitroquinoline-N-oxide(4NQO). (Leninger (1972) Biochemistry, Worth Publishers Inc., NY;Jeffrey H. Miller (1972) “Experiments in Molecular Genetics. Cold SpringHarbor Laboratory, Cold Springs Harbor, N.Y.). Preferred methods,include UV and NG. By way of example, mutant strains of Chrysosporiumcapable of producing increased levels cellulase compositions exhibitingenzymatic activity at neutral and or alkaline pH's may be generated byUV mutagenesis. By way of examples mutants may be produced by exposureof the fungal spores to UV light for a period of about 10 to about 180seconds, preferably 45 to about 90 seconds, and most preferably 65 to 75seconds. Mutageneses involving NG may involve varying the concentrationof the NG and exposure time. By way of example NG, at about 13milligrams/liter (mg/L) to about 400 mg/L of NG may be used for a periodof exposure of about 15 minutes to about 120 minutes. Alternativemethods for generating mutants include techniques in the field ofmolecular biology. Examples of such techniques include, but are notlimited to, oligonucleotide directed mutagenesis, linker scanningmutations or oligonucleotide directed mutagenesis using polymerase chainreaction (Ausubel (1987) Current Protocols in Molecular Biology).

Screening and selection of mutant Chrysosporium clones exhibitingimproved cellulase production can also be performed by conventionalmethodology. Examples of selection criteria for screening mutantsincludes, but is not limited, to the ability of the fungal colony tobreakdown cellulase as evidenced by cellulose clearing by the fungigrown on media plates containing cellulose and increased growth rate onmedia containing cellulose. By way of example, after mutagenesis thefungal sample may be plated on agar plates containing cellulose byconventional methodology. Colonies exhibiting increased growth andcellulose clearing (as is evidenced by clearing of the media surroundingthe isolate) may be selected. However, any other assay for celluloseclearing may be used. Once a mutant is isolated the stock or sporesderived from the mutant may be maintained by conventional methodology.The mutants isolated by this method may be cultured or fermented underthe conditions described herein and above for the wild type strains ofChrysosporium. The individual enzymatic components of the cellulasecomposition produced by the mutants may be partially sequenced ormicrosequenced to design synthetic to isolate the genes encoding theenzymatic proteins of interest. Thus, this invention relates to thenucleic acid sequences encoding the cellulase enzymes produced by theChrysosporium mutants and to the enzymes themselves. In yet anotherembodiment of this invention, the mutants isolated may be subjected tofurther mutagenisis and screened by the methods described herein above.

The present invention also relates to the production, isolation andpurification of cellulase enzymes, having both endoglucanase and/orcellobiohydrolase activity from Chrysosporium organisms of any speciesand strain desired and wherein the purification methodology is notlimited to the species of organism. Such purified, or partiallypurified, protein fractions and enzymes prepared therefrom are, inaccordance with the present invention, highly useful in applicationssuch as stone washing, color brightening, depilling and fabricsoftening, as well as other applications well known in the art. Suchpurified enzyme preparations are readily amenable to use as additives indetergent and other media used for such applications. These and othermethods of use will readily suggest themselves to those of skill in theart and need no detailed description herein. However, description oftypical methods of employing these preparations is provided in theexamples that follow.

All books, patents or articles references herein are incorporated byreference. The following examples illustrate various aspects of theinvention but in no way are included to limit the invention. Allpercentages are by weight and all solvent mixture propoitions are byvolume unless otherwise noted.

Materials and Methods

Enzyme assays. The assay for CMCase, used carboxymethyl cellulose as theenzymatic substrate, measured the initial rate of hydrolysis, andquantified the amount of reducing sugars released according to themethod of Somogyi and Nelson. The method is described in Methods inEnzymology, Vol. 160A, pp. 102-104. Endo-1,4-β-glucanase activity wasassayed viscometrically as a rate of the decrease of viscosity ofsoluble substrate CMC according to Bioorganicheskaya Khimia, Vol. 6, pp.1225-1242 (endoviscometric assay). Filter paper activity (FPA) or totalcellulase activity used filter paper as the substrate and estimated theactivity required to release 2 mg glucose from a 50-mg sample of filterpaper. The assay is based on the Commission on Biotechnology (IUPAC) forthe measurement for total cellulase activity or true cellulase and isdescribed in Methods in Enzymology, Vol. 160A, pp. 94-97. Avicelaseactivity was estimated as initial rate of reducing sugar formationduring hydrolysis if Avicell-cellulose (as described in BioresourceTechnology, Vol. 52, pp. 119-124). The assay of cellobiase usedcellobiose as the substrate and measured the amount of glucose released(described in Methods in Enzymology, Vol. 160A, pp. 111-112). The assayof β-glucosidase activity used p-nitrophenyl-β-D-glucoside as thesubstrate (described in Methods in Enzymology, Vol. 160A, pp. 109-1100).The protein was determined by Lowry method (according to Journal ofBiological Chemistry, Vol. 193, pp. 165-175). The assay of RBB-CMCase isbased on determination of the dye release from soluble substrate RBB-CMC(CMC, dyed with Remazolbrilliant Blue), reference to assay—see Megazyme(Australia) Ltd., Product Information Bulletin, April, 1995.Endo-cellulase can also be measured using the Cellazyme assay withAzurine-crosslinked HE-cellulose as substrate (see Megazyme ProductBulletin CEL, January, 1996).

EXAMPLE 1 Isolation of C1 Strain

The strain was isolated from samples of forest alkaline soil from SolaLake, Far East of Russian Federation (Pacific Coast of Russia, about5000 miles east from Moscow). A mixed soil sample was collected from 10different sites. One gram of each sample was transferred into a flaskwith 100 ml sterile tap water and sonicated with an ultrasonid dispenserfor 1 minute (0.44 Amp, 22 KHz). The suspension (diluted 1:500) wasinoculated into petri dishes with Czapek medium (pH 5.5-6.0) containing100 mg/L streptomycin. The study was conducted in three replicates.Colonies of various color shape and size were identified for a secondisolation step. Further isolation of the sample was performed on plateswith Czapek media, malt agar, potato dextrose agar, or Getchinson salinemedium pH 7.5 (Table 2). Plates were incubated at about 28° C. forseveral days. Selection for cellulase producers was performed oncellulose agar plates which contained the components shown in Table 1.Preparation of amorphous cellulose is described in Methods in Enzymologyvol. 160A.

TABLE 1 Cellulose agar plates Ingredients g/L KH₂PO₄ 1 KCl 0.1MgSO₄•7H₂O 0.3 NaCl 0.1 FeCl₃ 0.01 NaNO₃ 2.5 Amorphous cellulose 5 Agar15 pH 7.5

The plates were incubated for 3-7 days at 28° C. The formation of lightclearing halos around the colonies indicated cellulase activity. Onestrain, designated herein as C1, that exhibited significant levels ofcellulase activity was chosen for additional study. The strain wasdeposited at the All-Russian Collection of Microorganisms of RussianAcademy of Sciences, (VKM), abbreviation in English—RCM), BakhrushinaSt. 8: Moscow, Russia, 113184 under the Budapest Treat on Aug. 29, 1996,as Chrysosporium lucknowense Garg 27K, VKM-F 3500 D).

EXAMPLE 2 Characterization of C1 Strain

Growth of the C1 strain on potato dextrose agar gives colonies of 55-60mm diameter after 7 days. C1 colonies exhibit a white-cream color, thesurface is velvet-like and has a slightly raised center. The edge of thecolonies is a flat, thin and fibereil. The back side of the colonies hasa light cream color.

The mycelium has hyaline and is slightly branched and smooth. The hyphaeare thin-walled. Air hyphae are septate and form spores of 2.0-3.0micrometers width; the substrate hyphae are sterile.

The conidia are terminal and lateral. No intercalary conidia were found.The majority of conidia are connected with hyphae through short stems orshort side branches. The conidia are separated but adjacent. Conidia arehyaline, thin-walled, oval or clavate, and single cellular. Their sizevaries from 4 to 10 micrometers in diameter.

The C1 strain can be maintained on malt extract agar (at 4° C.), andtransferred each six months. Maintenance in liquid nitrogen and bylyophilization is also possible. The C1 strain is haploid, filamentous,can grow on agar plates with growth restricting agents like bovinebile(1.5%), and produces spores.

EXAMPLE 3 Classification of C1 Strain

According to Sutton classification (Van Dorschot, C.A.N. [1980] “Arevision of Chrysosporium and allied genera,” in Studies in Mycology,No. 20, Centraaddbureau voor Schimmelcultures, Baarn, The Netherlands,pp. 1-36), the C1 strain of the subject invention belongs to the orderof Hyphomycetales, family of Moniliaceae, genus of Chrysosporium,species of Chrysosporium lucknowense Garg 1966. This classification wasbased on observation of the following characteristics of the C1 strain:

1. Signs of Hyphomycetales order. Conidia are produced directly onmycelium, on separate sporogenous cells or on distinct conidiophores.

2. Signs of Moniliaceae family. Both conidia and conidiophores (ifpresent) are hyaline or brightly colored; conidiophores are single or inloose clusters.

3. Signs of Chrysosporium Corda 1833 genus. Colonies are usuallyspreading, white, sometimes cream-colored, pale brown or yellow, feltyand/or powdery. Hyphae are mostly hyaline and smooth-walled, withirregular, more or less orthotopic branching. Fertile hyphae exhibitlittle or no differentiation. Conidia are terminal and lateral, thallic,borne all over the hyphae, sessile or on short protrusions or sidebranches, subhyaline or pale yellow, thin- or thick-walled, subglobose,clavate, pyriform, orobovoid, 1-celled, rarely 2-celled, truncate.Intercalary conidia are sometimes present, are solitary, occasionallycatenate, subhyaline or pale yellow, broader than the supporting hyphae,normally 1-celled, truncate at both ends. Chlamydospores areoccasionally present.

4. Signs of Chlysosporium lucknowense Garg 1966 species. Colonies attain55 mm diameter on Sabouraud glucose agar in 14 days, are cream-colored,felty and fluffy; dense and 3-5 mm high; margins are defined, regular,and fimbriate; reverse pale yellow to cream-colored. Hyphae are hyaline,smooth- and thin-walled, little branched. Aerial hyphae are mostlyfertile and closely septate, about 1-3.5 mm wide. Submerged hyphae areinfertile, about 1-4.5 mm wide, with the thinner hyphae often beingcontorted. Conidia are terminal and lateral, mostly sessile or on short,frequently conical protrusions or short side branches. Conidia aresolitary but in close proximity to one another, 1-4 conidia developingon one hyphal cell, subhyaline, fairly thin- and smooth-walled, mostlysubglobose, also clavate orobovoid, 1-celled, 2.5-11×1.5-6 mm, withbroad basal scars (1-2 mm). Intercalary conidia are absent.Chlamydospores are absent.

5. Description of C1 strain. Colonies grow to about 55-60 mm diameter insize on potato-dextrose agar in about 7 days; are white-cream-colored,felty, 2-3 mm high at the center; margins are defined, regular,fimbriate; reverse pale, cream-colored. Hyphae are hyaline, smooth- andthin-walled, little branched. Aerial hyphae are fertile, septate, 2-3 mmwide. Submerged hyphae are infertile. Conidia are terminal and lateral;sessile or on short side branches; absent; solitary, but in closeproximity to one another, hyaline, thin- and smooth-walled, subglobose,clavate or obovoid, 1-celled, 4-10 mm. Chlamydospores are absent.Intercalary conidia are absent.

Conclusion. C1 is a strain of Chrysosporium lucknowense Garg 1966. Forconvenience the cellulase made by this strain is referred to herein as“C1” or “C1 cellulase.”

EXAMPLE 4 Assay for Cellulase Activity

The C1 strain was grown in 800 ml shake flasks rotated at 220 rpm andincubated at 28° C. The C1 strain was grown in saline Getchinson medium(See Table 2) (pH 7.5) containing 5 g/L of various nutrients, and insome cases with 2 g/L microcrystalline cellulose. One hundred ml ofmedia were added to each flask.

TABLE 2 Getchinson medium for shake flasks g/L KH₂PO₄ 1 KCl 0.1MgSO₄•7H₂O 0.3 NaCl 0.1 FeCl₃ 0.01 NaNO₃ 2.5

Combinations of glucose and microcrystalline cellulose, dextrose andmicrocrystalline cellulose, glycerol and microcrystalline cellulose,lactose and microcrystalline cellulose resulted in very low growth,formation of large aggregates of mycelium, and in the absence ofcellulase activities (CMCase assay). The results are presented in Table3. Additions of nitrogen organic sources, i.e., peptone, corn steepliquor, or yeast extract enhanced growth and cellulase production anddid not result in mycelium aggregates.

Lactose and yeast extract gave the highest cellulase production by C1.Similar results were obtained when the lactose and yeast extract weresubstituted with 25 g/L sweet beet pulp, 15 g/L barley malt, and 5 g/Lwheat bran.

TABLE 3 Effect of carbon and nitrogen sources on CMCase activity of C1(shake flasks results) CMCase activity (units/ml) at pH 7.0 DaysSubstrate 3 5 7 Glucose + cellulose 0 0 0 Dextrose + cellulose 0 0 0Glycerol + cellulose 0 0 0 Lactose + cellulose 0 0 0 Lactose + cornsteep liquor 0 0 0.9 Lactose + peptone 10.7 7.4 14.8 Lactose + yeastextract 0 18.5 10.0 Cellulose + peptone 0.3 1.2 1.6 Cellulose + cornsteep liquor 1.9 2.8 5.5

EXAMPLE 5 Production of Cellulase for Stone Wash Tests

1. Production in shake flasks. C1 strain was grown in 800 ml shakeflasks rotated at 220 rpm and incubated at 28° C. for seven days. Thegrowth medium 100 ml per flask was saline Getchinson medium (see Table2) (pH 7.5) containing 25 g/L sweet beet pulp, 15 g/L barley malt, and 5g/L wheat bran. The cell mass was separated by centrifugation and thecell-free supernatant was lyophilized and stored for further tests. C1cellulase preparation #s 47.1.1 to 47.15.1 were produced in this manner.C1 preparation #47.16.1 was produced by the same manner, but cell-freesupernatant after centrifugation was ultrafiltrated using a 10 kDacutoff membrane before lyophilization. C1 preparation #'s 47.18.1 to47.22.1 were produced by the same manner in shake flasks with Getchinsonmedium, but containing lactose (0.5% w/v) and peptone (0.5% w/v) insteadof sweet beet pulp, barley malt and wheat bran. The cell mass wasseparated by centrifugation and the cell free supernatant waslyophilized and stored for further tests. Preparation #'s 47.1000,47.1001, 47.2000 & 47.2001 were produced in shake flasks by the samemanner as preparation ft's 47.1.1-47.15.1 except that they were producedusing other Chrysosporium strains. Specifically, 47.2001 was produced byChrysosporium pannorum, preparation 47.2000 was produced byChrysosporium pruinosum, preparation 47.1001 was produced byChrysosporium keratinophilim and preparation 47.1000 was produced byChrysosporium queenslandicum (see Example 8). The protein content andactivity fingerprints of these C1 preparations are shown in Table 4.

TABLE 4 Protein content and activity fingerprints of C1 preparations andpreparation #'s 47.1000, 47.1001, 47.2000 & 47.2001 which were preparedfrom other species of Chrysosporium sp. CMC- Endo Avice- β-Gluco-Preparation Protein, FPA, ase, (visc), lase, sidase, # % FPU/g U/g U/gU/g U/g 47.1.1 22 13 170 120 23 135 47.2.1 26 14 137 110 22 190 47.3.115 19 140 128 18 198 47.4.1 18 23 150 133 55 220 47.5.1 16 20 179 120 71185 47.6.1 17 22 224 134 82 280 47.7.1 8 4 78 123 10 22 47.8.1 22 14 168123 19 124 47.9.1 28 15 204 174 23 151 47.10.1 24 11 181 185 16 14747.11.1 28 16 234 191 25 269 47.12.1 26 14 167 138 20 178 47.13.1 25 9137 110 13 141 47.14.1 15 6 39 33 9 59 47.15.1 14 6 95 44 10 75 47.16.116 10 146 39 15 107 47.17.1 7 3 100 34 5 29 47.18.1 10 30 120 38 10 4247.19.1 14 4 28 10 4 11 47.20.1 14 6 17 5 1 9 47.21.1 13 3 34 5 3 947.22.1 14 5 35 6 3 10 47.1000 18 4 31 35 6 89 47.1001 13 6 103 38 10 6647.2000 10 3 78 31 7 67 47.2001 13 3 45 39 7 4 47.0325 50 155 4965 964184 248 47.0528 67 111 13500 1782 232 423

2. Production in fermentors. C1 cellulase was produced in a 10-L“ANKUM-1M” fermentor with Getchinson medium, lactose (0.5% w/v), peptone(0.5% w/v), and chloramphenicol (50 mg/mL). Initial volume of thenutrition medium was 7.0 L, final volume after fermentation was 7.3 L.The dissolved oxygen concentration (DO), agitation speed, aerationlevel, temperature, and pH were controlled. Fermentation was carried outas a batch-mode. The temperature of the fermentation was controlled at28° C. The initial pH was 7.5 and was later maintained at that level byaddition of NH₄OH (12% w/v). The aeration was at 4-5 L/minute andagitation at 400-500 rpm. The DO was maintained at above 50%. Samples(30 ml) were taken for analysis every 8 hours. At the end offermentation, fungal biomass was separated by centrifugation (10,000 g,room temperature, 20 minutes), and culture filtrate was lyophilized andstored for further tests. The results are shown in Table 5. Cellulasepreparation #47.17.1 was produced in this manner. Protein content andactivity fingerprint of this C1 preparation is shown in Table 4.

TABLE 5 Production of C1 cellulase in 10-L fermentor Time (h) DO (%)Reducing sugars (g/L) CMCase (U/mL) 0 100 4.8 0 8 90 4.7 0 16 54 4.4 024 66 1.2 4 32 70 0.4 10 40 73 0.3 11.5 48 70 0.1 5 56 70 0 1

3. Production of C1 Preparation #s 47.0325 and 47.0528. C1 cellulasepreparation #47.0325 was produced using the wild type C1 strain,preparation #47.0528 was produced using an improved mutant obtained fromthe wild type C1 strain. These preparations were grown up fermentorsunder the conditions described in Examples 13 and 15. Preparation47.0325 was produced using a batch fermentation and 47.0528 was producedusing a fed batch fermentation protocol.

4. Preparation of Humicola wild type preparation #'s 14.22.1 & 14.23.1The wild type Humicola grisea var. thermoidea preparation #14.22.1 wasproduced from the ATCC 16453 strain and the wild type Humicola insolenspreparation #14.23.1 was produced from the ATCC 16454 strain. TheseHumicola wild type preparations were produced in shake flasks using thesame method as described above for (Production in shake flasks) of C1preparation #'s 47.1.1-#47.15.1.

EXAMPLE 6 Comparison of C1 to Other Neutral Cellulases

The FPA, CMCase and endoglucanase activities of C1 enzyme preparation#47.0528 were compared to commercial Humicola insolens (Denimax XT) andto wild ATCC-type Humicola (preparation #'s 14.22.1 Humicola grisea var.thermoidea (ATCC 16453) & 14.23.1 Humicola insolens (ATCC 16454) neutralcellulases. The results are given in the Table 6. The total activitiesof C-1 #47.0528 are clearly higher than those of neutral cellulases fromwild type Humicola and from commercial Humicola insolens preparation.The specific CMCase and endoglucanase activities (as units per gram ofdry preparation or units per gram of protein) of C-1 47.0528 are higherthan those of all tested Humicola preparations listed in Table 6. Thespecific FPA of C-1 #47.0528 is higher than the specific FPA of Humicolawild type preparations #14.22.1 & 14.23 and slightly lower than thespecific FPA of the Humicola insolens commercial product Denimax XT. ThepH and thermal stability of C1 cellulase was similar to Denimax XT.

TABLE 6 Comparison of C1 and Humicola cellulases. Endo Endo FPA CMCase(visc) FPA CMCase (visc) unit/1 gram of units/1 gram of Protein % drypreparation protein C1 (47.0528) 67 111 13,500 1782 165 20,115 2,655Humicola sp. (# 14.23.1) 10 2 28 30 20 280 300 Humicola sp. (# 14.23.1)10 1 11 19 10 110 190 Denimax XT 13 25 450 99 192 3,460 761 (commercial)(*) Activities were measured at pH 5.0 and 50° C.

EXAMPLE 7 The Effect of pH and Temperature on Activity and Stability ofC1 FPA and CMCase Activities

The FPA and CMCase activities of C1 exhibit optimal stability andactivity at about pH 6-7 and about 50-60° C.; the pH optimum for CMCaseactivity is about 6.5, and the optimum temperature is about 55° C. (seeTables 8,9). At pH 8.0 (50° C.), CMCase possesses 80% activity, andFPA—78% activity, at pH 9.0 (50° C.), CMCase possesses 65% activity, andFPA—52% activity (see Table 7.).

TABLE 7 the effect of pH on FPA and CMCase activities of C1 cellulase(#47.19.1) at 50° C. pH (50° C.) FPA (%) CMCase (%) 4.0 50 60 4.5 68 705.0 75 78 5.5 80 80 6.0 92 90 6.5 100 100 7.0 95 95 7.5 90 92 8.0 78 808.5 60 75 9.0 52 65

The incubation time for the FPA assay was 60 minutes, the incubationtime for CMCase assay was 5 minutes.

TABLE 8 The effect of temperature on FPA and CMCase activities of C1cellulase (#47.19.1), at pH 7.0 Temperature (C.) FPA (%) CMCase (%) 4045 50 45 60 55 50 70 65 55 100 100 60 70 60 65 40 30 70 20 25

The incubation time for the FPA assay was 60 minutes, the incubationtime for the CMCase assay was 5 minutes.

TABLE 9 Stability of CMCase of C1 cellulase (# 47.19.1) at 50° C. CMCaseactivity remained (%) Time (h) pH 5.1 pH 7.2 pH 7.7 pH 8.5 0 100 100 100100 0.5 100 98 95 85 1 100 95 93 55 2 100 82 78 32 3 100 78 65 25 5 10075 45 15

The CMCase of C1 exhibits high stability at optimal pH and temperature:For Example; at pH 7.2 and 50 C CMCase possesses 95% activity after 1hour and 75% activity after 5 hours, at pH 7.7 and 50 C CMCase possesses93% activity after 1 hour and 45% activity after 5 hours (See Table 9.).

EXAMPLE 8 Neutral and or Alkaline Cellulase Activity/PerformanceDemonstrated in Other Strains of the Same Genera of Chrysosporium

Various strains of the Chrysosporium genus were tested for cellulaseproduction. The full names and origins of these strains are describedbelow.

Strains obtained from the American Type Culture Collection (ATCC).Rockville. Md., include:

1. ATCC 44006 Chrysosporium lucknowense

2. ATCC 34151 Chrysosporium pannorum

3. ATCC 24782 Chrysosporium pruinosum

Strains obtained from the Russian Collection of Microorganisms (VKM)include:

1. VKMF-2119 Chrysosporium keratinophilum

2. VKMF-2875 Chrysosporium keratinophilum

3. VKMF-2120 Chrysosporium lobatum

4. VKMF-2121 Chrysosporium merdarium

5. VKMF-2116 Chrysosporium queenslandicum

6. VKMF-2117 Chrysosporium queenslandicum

7. VKMF-2877 Chrysosporium tropicum

Two types of growth media were used in this study: medium A—Getchinsonwith sugar beet press, barley malt, and wheat bran: and mediumB—Getchinson with peptone and lactose. The compositions of the media aredescribed in Table 11.

TABLE 11 Media for flasks studies Medium A g/L Medium B g/L K₂HPO₄ 1K₂HPO₄ 1 KCl 0.1 KCl 0.1 MgSO₄•7H₂O 0.3 MgSO₄•7H₂O 0.3 NaCl 0.1 NaCl 0.1FeCl₃ 0.01 FeCl₃ 0.01 NaNO₃ 2.5 NaNO₃ 2.5 Sweet beet pulp 25 Lactose 5Barley malt 15 Peptone 5 Wheat bran 5 pH 7.5 pH 7.5

The strains were grown in shake flasks at 220 rpm and at 28° C. Samplesof each strain grown in Medium A were taken for analysis after 6 and 7days of culture. Samples of strains grown in Medium B were taken after 5days in culture. All samples were assayed for CMCase activity at pH 5and 7. The results of the CMCase assay are shown in Table 12.

TABLE 12 Cellulase production by different strains of Chrysosporiummedium A medium A medium B (6 days) (7 days) (5 days) CMCase CMCaseCMCase Strains RS pH 5 pH 7 RS pH 5 pH 7 RS pH 5 pH 7 1 VKMF 2.7 0 0.462.6 0.00 0.00 2.3 0.21 0.09 2117 2. VKMF 1.1 0.22 0.04 0.2 0.38 0.61 4.00.58 0.59 2116 3. VKMF 1.9 0 0.57 1.1 0.25 0.10 2.5 0.25 0.09 2121 4.ATCC 3.4 0.33 1.40 1.9 1.85 0.11 3.0 1.10 0.06 24782 5. ATCC 1.0 1.540.90 0.9 0.17 0.20 4.3 0.81 0.90 34151 6. ATCC 4.4 0.21 0.49 2.0 0.680.34 2.5 1.29 0.06 44006 7. VKMF 4.1 0 0.08 2.7 0.29 0.00 3.8 0.95 0.042119 8. VKMF 4.5 0 0.17 2.3 0.23 0.00 2.3 0.12 0.00 2120 9. VKMF 1.6 01.01 1.7 0.00 0.00 3.8 1.96 0.05 2875 10. VKMF 2.4 0 0.03 0.8 0.22 0.005.0 0.43 0.00 2877 11. C1 2.9 1.70 1.65 nt nt nt 0.1 0.89 0.80 (VKMF3500D) RS = concentration of reducing sugars in the fermentation mediumat the end of fermentation, g/L (Nelson-Somogyi method). pH 5, pH 7 =the values of pH under which the CMCase activity of the fermentationbroth was assayed. CMCase activity in U/ml. nt = not tested

In the cases of strains ATCC 34151 Chrysosporium pannorum, ATCC 24782Chrysosporium pruinosum, VKMF-2875 Chrysosporium keratinophilum, VKMF2116 Chrysosporium queenslandicum the cell mass was separated bycentrifugation and cell free supernatant concentrated from 5 liters to0.5 liter by ultrafiltration using 10 kDa cut-off membrane. Then theultrafiltrated concentrate was lyophilized and stored for tests.

The following #-s of cellulase dry preparations were used:

47.2001—ATCC 34151 Chrysosporium pannorum,

47.2000—ATCC 24782 Chrysosporium pruinosum,

47.1001—VKMF-2875 Chrysosporium keratinophilum.

47.1000—VKMF 2116 Chrysosporium queenslaandicum.

Protein content and activity fingerprints of these preparations aregiven in Table 4.

EXAMPLE 9 Stone Wash Tests

A. Tests with 2-L special washing machine. This system assesses thestone wash performance characteristics related to abrasion andbackstaining using only small amounts of enzyme.

Desizing. Forty pieces (30 g each, 25×20 cm) of denim fabric (roll) (1.2kg) were desized in a household washer at 60° C. for 20 minutes using afabric:liquor ratio of 1:6 (7.2 L) and 0.5 g/L (3.6 g) Sandoclean PCliquid (nonionic washing and wetting agent on base of ethyoxylated fattyalcohols with an average of 6 moles of ethylene oxide, 1 g/L (7.2 g)Sirrix 2UD (acidic buffered sequestration) and 1 g/L (7.2 g) Bactosol TKliquid (high temperature stable alpha-amylase) at a pH of about 5 to 6.After 20 minutes, the liquor was drained and the pieces washed for 5minutes with cold water (14 L) liquid ratio 1:10. The pieces were driedat 40° C. and used as a stock of comparable samples for thedetermination of cellulase activity

The cellulase treatment of the garment pieces was carried out in awashing machine consisting of an inner drum of 29 cm diameter drum—10.6l total volume (drum rotates at 20 rpm—five turns left—five turnsright). Each piece of fabric was sewn together with 4 rubber stoppersprior to the cellulase treatment to give a garment package that ensuredthat the mechanical effect occurred mainly on the darker outer side ofthe garment. Each drum was filled with one package and 10 additionalrubber stoppers.

The general wash conditions were: 30 g desized denim jean fabric,cellulase in 0.02 M citrate buffer, 50° C., 60 minutes, garment:liquorratio 1:4. After the cellulase treatment the package was washed with hotwater (50 C) (garment:liquid ratio 1:20) for 5 minutes and dried forevaluation.

Application trials were conducted using various C1 cellulasepreparations along with other cellulase preparations prepared fromdifferent species of Chrysosporium as well as the commercial NovoNordisk neutral cellulase products, Denimax XT (U.S. Pat. No. 4,435,307)and Ultra MG (WPO 91/17243). These application trials were set up toevaluate the stone wash performance characteristics of C-1 as well asseveral other species of Chrysosporium cellulases vs Novo's commercialneutral cellulases. The trials were run at neutral and alkaline pH's(6.5, 6.7, 7.0, and 8.0). The results are presented in Table 13.Garments treated with various C1 and other Chrysosporium cellulasepreparations showed similar wash performance characteristics to those ofthe commercial neutral cellulases Denimax XT and Denimax Ultra MG. TheC-1 and other Chrysosporium cellulase preparations showed good softeningeffect, bleaching/overall shade reduction, abrasion levels as well aslow backstaining values when run under neutral and alkaline pHconditions. Datacolor measurement is based on the degree of lightness ofthe sample (reflectance). The sample is exposed to white light (2 pulsedXenon flash lamps) and the remission is detected between 400 and 700 nmwith 16 diodes. Reflectance from the front side, the higher value themore abrasion. Reflectance from the back side, the higher value the morebackstaining.

TABLE 13 Enzyme Wash With Special 2 Liter Machine (135 grams of denimper run) Endo CMCase (visc) Liquor Time Datacolor Datacolor EnzymeAmount g % OWG U/g /run U/g /run ° C. ratio pH (min) Buffer AbrasionBackstng C-1 1.995 1.5 234 474 191 381 50 1:11 7.2 60 0.02MP 13.1 1.847.11.1 Denimax XT 0.133 0.10 450 60 99 13 50 1:11 7.0 60 0.02MP 13.12.4 C-1 2.100 1.5 167 338 138 290 50 1:11 6.7 60 0.02MP 14.2 1.8 47.12.1Denimax XT 0.420 0.30 450 182 99 42 50 1:11 6.6 60 0.02MP 14.0 2.3 C-147.9.1 3.29 2.44 204 671 174 572 50 1:11 6.7 60 0.02MP 17.1 1.8 C-1 2.31.7 146 336 39 90 50 1:11 6.7 60 0.02MP 16.2 2.3 47.16.1U 47/1000.1 7.05.19 48 336 14 98 50 1:11 6.5 60 0.02MP 12.9 2.1 47/2001.1 7.15 5.30 47336 20 143 50 1:11 6.5 60 0.02MP 14.7 1.7 Denimax 0.132 0.10 134 18 24332 50 1:11 7.3 60 0.02MP 14.1 3.5 UltraMG C-1 7.14 5.29 28 200 9 64 501:11 6.5 60 0.02MP 14.7 1.9 47.19.1 C-1 0.068 0.05 4965 338 964 66 501:11 7.0 60 0.02MP 15.1 2.3 47.0325 Denimax XT 1.0 0.74 450 450 99 99 501:11 6.5 60 0.02MP 19.1 2.8 C-1 0.08 0.05 4800 384 1782 143 50 1:11 6.560 0.02MP 18.5 3.0 47.0528 C-1 0.136 0.10 4965 675 964 131 50 1:11 6.060 0.02MP 18.3 3.8 47.0325 C-1 0.136 0.10 4965 675 964 131 50 1:11 7.060 0.02MP 18.9 3.2 47.0325 C-1 0.136 0.10 4965 675 964 131 50 1:11 8.060 0.02MP 16.7 2.5 47.0325 Humicola 9.18 6.80 28 257 30 275 50 1:11 6.760 0.02MP 14.9 1.3 14.22.1 Humicola 9.18 6.80 11 101 19 174 50 1:11 6.760 0.02MP 12.5 1.5 14.23.1 T. reesei 0.30 0.22 9190 2737 2000 600 501:11 4.8 60 0.02CA 17.5 8.0 CP Blank 0.00 0.00 0 0 0 0 50 1:11 6.5 600.02MP 9.1 n/a 0.02MP = Phosphate Buffer System 0.01CA = Citric AcidBuffer System T. reesei CP = Commercial acid cellulase product producedfrom Trichoderma reesei. Datacolor Abrasion = reflectance from the frontside, the higher the values, the more abrasion, Blank = 9.1 DatacolorBackstng = reflectance from the back side, the lower the values, thelower the back staining % OWG = for example for 1% OWG, 1 lb of enzymeis used on 100 lbs of garment

B. Tests with 35 lb washing machine. Application Trials were run in a 35lb washing machine (35 lb washing machine brand is Milnor—washer RPM is30). Load size is 2400 g (3 garments), garments used are Levi's 505jeans. Water level for cellulase bath is 15 L for a liquor ratio of6.25:1 (low). The water level for all other baths is 24 L for a liquorratio of 10:1 (Med). The buffering system used is MAP—monoammoniumphosphate and DAP—diammonium phosphate to maintain the pH of 6.7 duringthe cellulase bath. In Trials 4, 5, 6 & 7 a nonionic detergent was addedto the cellulase bath, it is known that adding a detergent to thecellulase bath will help in reducing the backstaining on the garments.Zeke is a desizing product. SSCE is Superscour, a nonionic detergent(Zeke and Super Scour are commercial specialty textile chemical productsoffered by CPN International, Ltd., Inc of Jupiter, Fla.). One Exampleof the Wash Formulas used in these trials is Trial 2. below;

Wash Formula - Trial 2. (C-1 47.0528) Load (g) 2400 (3 gmts) Fabric:Denim Formula Time: 1:30 Machine: 35# Milnor Weight: 14.5 oz Developedby: Step Operation Time (min) Level Temp (F.) Chemical Amount % OWG pH 1Desize 10 Med 150 Zeke 48 g 2 2 Drain Balance 3 Rinse 2 Med 140 4 DrainBalance 5 Rinse 2 Med 130 6 Drain Balance 7 Abrasion 75 Low 125 MAP 29 gbuffer 6.7 (15L) DAP 10 g C-1 1.2 g  0.05 8 Drain Balance 9 Wash 10 Med160 SSCE 24 g 1 10 Drain Balance 11 Rinse 3 Med 120 12 Drain Balance 13Rinse 3 Med 100 14 Drain Balance 15 Rinse 3 Med 100 16 Drain Balance 17Extract 2

In the example above, and in commercial use, one skilled in the art willappreciate that the use of pumice stones in the stonewash process willenhance the overall stonewash effect on the garments.

The results in Table 14. show that the C1 cellulase preparations#47.0325 and #47.0528 performed better in terms of the overall level ofabrasion achieved on the garments and well within the range of thebackstaining level of the other commercial neutral cellulase productstested.

TABLE 14 Comparison of C-1 Cellulase Preparations 47.0325 & 47.0528 ToCommercial Neutral Cellulases Denimax XT & BTU 202-318 (which containsDenimax XT) % T Trial Cellulase OWG Wt (g) Detergent (° F.) pH t (min) 1Denimax XT 0.50 12.0 no 130 6.7 75 2 C-1 47.0528 0.05 1.2 no 125 6.7 753 C-1 47.0325 0.10 2.4 no 125 6.7 75 4 Denimax XT 0.50 12.0 yes 130 6.775 5 C-1 47.0528 0.05 1.2 yes 125 6.7 75 6 C-1.47 0325 0.10 2.4 yes 1256.7 75 7 BTU 202-318 2.50 60.0 yes 130 SB 75 ABRASION BACKSTAINING (Mostto Least) (Least to Most) Trial 5 Trial 4 Trial 6 Trial 5 Trial 2 Trial6 Trial 3 Trial 3 Trial 4 Trial 1 Trial 1 Trial 2 Trial 7 Trial 7 FigureLegand for Table 14 All of the trials in table 14. were cleaned up withSuper Scour (nonionic detergent) at 1.0% OWG, 160 F. for 5 minutes. SB =Self Buffered - the commercial product “ROCKSOFT” BTU 202-318 containsDenimax XT, detergent and a buffer system as well as other additives tohelp enhance the stone wash performance of this commercial product.

C. Tests with 60-L special washing machine. Whole denim garments weredesized as described for the 2-L washing machine tests. Each wash testwas made with 1 pair of jeans (700 g), 2.8 L liquid (fabric:liquid ratio1:4). All jeans were from the same dye lot. They were prewashed using anoxidation method for 15 minutes, then dried. Blue jeans washed atneutral pH with formulated C1 cellulase preparations 47.0325 using 2.4grams per trial and 47.0528 using 1.5 grams for one trial and using 1.0gram for a second trial were compared directly against blue jeans washedunder neutral pH conditions and similar formulations using Denimax XT at12 grams per trial and two other commercial neutral cellulases; BactosolJE using 2.0% OWG and BTU 202-318 using 2.0% OWG (Bactosol JE and BTU202-318 contain Denimax XT, buffer, detergent as well as other additivesto enhance their wash performance). Table 15. shows that the blue jeansfrom all three C-1 trials outperformed the three commercial neutralcellulase products in terms of the level of abrasion achieved as well asthe overall color reduction of the garments. The level of backstainingon the blue jeans from all six trials was very good, they were verysimilar to one another and what one would expect and see when usingNovo's neutral cellulase Denimax XT. The backstaining values for allthree of these C-1 trials were within the range of the backstainingvalues as shown in Table 13. The finished garments from these trials andthe trials as rated and shown in Table 14 above were rated in a blindstudy by four independent groups, of three or more people per group. Thepeople that made up each of these groups are considered to be skilled inthe art of stonewashing. They were asked to place each of the garmentsin the following order: (1) Greatest overall abrasion and colorreduction to least overall abrasion and color reduction; and (2)Backstaining, lowest level of backstaining to highest level ofbackstaining (See Table 15).

TABLE 15 ABRASION/ COLOR TRIAL ENZYME DOSAGE BUFFER DETERGENT REDUCTIONBACKSTAINING Trial A C-1 47.0528 1.5 grams Phosphate Yes ++++++ 5 TrialB C-1 47.0528 1.0 grams Phosphate Yes +++++ 2 Trial C C-1 47.0325 2.4grams Phosphate Yes +++++ 4 Trial D Denimax XT 12.0 grams Phosphate Yes++++ 3 Trial E BTU 202-318 2.0% OWG Phosphate Yes +++ 6 Trial F BactosolJE 2.0% OWG Citrate Yes +++ 1 Legend for Table 15: Abrasion/ColorReduction - ++++++ (+6) best (= > ++++ (4) is considered good and wascomparable to commercial netural cellulases (e.g. - Denimax XT)Backstaining - The lower number the better (all jeans were judged to bewithin the range of backstaining as found when using Novo's Denimax TX).Neutral cellulase significantly decreased backstaining compared withtraditional acid cellulases such as Trichoderma (see Example 13) % OWG—%Of Weight of Garment, for example for 100 lbs of jeans dryweight at 1%OWG, 1 lb of enzyme is used.

D. Light reflectance. Another test to evaluate backstaining is tomeasure the light reflectance of a treated fabric. At the end of washingtreatment, jeans samples were analyzed using a reflectometer at twodifferent wavelengths: (1) the higher the signal detected at 680 nm(measured at the outside of the jeans), the lower the backstaining; and(2) the higher the signal detected at 420 nm (measured at the inside ofthe jeans), the lower the backstaining. Table 16. compares thereflectance values of denim jeans after treatment with commercialcellulases from Novo Nordisk and Genencor International to C-1preparation #47.6.1.

TABLE 16 Enzyme 680 nm 420 nm Denimax L (neutral cellulase, Novo) 23 20Primafast 100 (acid cellulase, Genencor) 20 13 C1 47.6.1(neutral/alkaline cellulase) 22 18

The light reflectance values for the C1 cellulase were similar to thoseobtained with Novo Nordisk's commercial product Denimax L, a neutralcellulase, at both 680 and 420 nm and the light reflectance values forC1 cellulase were significantly better than those obtained withGenecor's commercial product Primafast 100, a acid cellulase, at both680 and 420 nm.

E. Tests in Semi-Industrial Washing Machine.

-   -   Test #1.    -   2 Jeans, weight 1343 gr    -   Water ratio 6:1    -   pH 5.5    -   Temp. 54° C.    -   Enzyme: C1 (preparation #47.6.1) 12 gr    -   (0.9%)    -   Abrasion time 90 minutes    -   Drop bath    -   Rinse 5 minutes with 1% non-ionic detergent at 66° C.    -   Drop bath    -   Rinse cold    -   Drop bath    -   Soften for 5 minutes with cationic softener at Extract and dry.

Test #2.The same procedure as Test #1, above, except Denimax 700 T (2%OWG 28.9 gr) enzyme was used and wash conditions were conducted at pH7.0, 54° C.

C1 cellulase was compared to Denimax 700 T, a neutral cellulasecommercial product made by Novo. All jeans were from the same dye lot.They were prewashed for 15 minutes using an oxidation method then dried.

The jeans treated with C1 cellulase preparation #47.6.1 showed slightlyless abrasion and lower backstaining than the jeans treated with Denimax700T cellulase.

EXAMPLE 10 C1 Cellulase as an Additive to Laundry Detergent

A. Soil Release from Cotton

Wash performance of C1 cellulase preparation #47.9.1 was tested usingthe wash-performance procedure PW 9406 (Solvay). Soil (ink) release fromcotton fabric was tested by Delta Reflectance (%). Wash test compared aC1 cellulase preparation (#47.9.1) to Celluzyme 0.7 T from Novo Nordiskin the presence and absence of alkaline protease Opticlean L500. Theresults of this test are shown in the Table 17.

C1 cellulase has soil release properties from ink soiled cotton atneutral pH in a color type detergent as the cellulase enzyme fromHumicola insolens.

TABLE 17 Detergent wash test with C1 cellulase (*) CMCase ReflectanceData (%) Enzyme tested (pH 7.0) dosage (U/I) 1 2 Cellulzyme 0.7 T 2003.68 4.75 Celluzyme 0.7 T 500 2.68 4.07 Celluzyme 0.7 T + 5000 DU/l (**)200 2.07 3.13 Celluzyme 0.7 T + 5000 DU/l (**) 500 2.08 3.22 C1 # 47.9.1200 2.18 2.88 C1 # 47.9.1. 500 2.77 3.72 C1 #47.9.1 + 5000 DU/l (**) 2001.15 1.91 C1 #47.9.1 + 5000 DU/l (**) 500 2.81 3.30 None (control) none0 0 AADU = Du = Delft unit, Du/l = Defft unit per liter (*) 40 C.°, 45min, drying at 68° C., 75 min (**) Alkaline protease Opticlean L500

B. The Stability of C1 Cellulase with Serine Proteases

As serine proteases, a trypsin (3.2 μM, from Bovine Pancreas, activity10,000-13,000 N-benzyl-L-argine ethylester (BAEE)/mg, Sigma T-8253) andan α-chymotrypsin (8 μM, from Bovine Pancreas, 40-60 U/mg, Sigma C-4129)were used.

The proteases were incubated with C1 cellulase at 20° C. and pH 7.0.Chymotrypsin did not decrease C1 activity for 12 hours and trypsin ledto a slight decrease (around 20%) of C1 activity, see Table 18.

Trypsin and chymotrypsin did not significantly change the stability ofC1 CMCase at pH-s 4.5 and 7.0 at 50° and 57° C., see Table 18.

TABLE 18 The effect of proteases on CMCase activity of C1 cellulase (#47.9.1) Temperature Incubation CMCase activity Protease (° C.) pH time(h) remaining (%) None (control) 20 7.0 12 70 +Chymotrypsin 20 7.0 12 70+Trypsin 20 7.0 12 50 None (control) 50 4.5 3 100 +Chymotrypsin 50 4.5 3100 +Trypsin 50 4.5 3 100 None (control) 50 7.0 3 78 +Chymotrypsin 507.0 3 60 +Trypsin 50 7.0 3 68 None (control) 57 4.5 3 62 +Chymotrtrypsin57 4.5 3 60 +Trypsin 57 4.5 3 62 None (control) 57 7.0 3 30+Chymotrypsin 57 7.0 3 30 +Trypsin 57 7.0 3 30

C. The Effect of Citrate, EDTA, Tween-80 and Persulfate on CMCaseActivity

Changing from acetate to citrate buffer (a chelating agent) did noteffect the of C1 CMCase activity (molarity of buffers—0.1 M, pH 4.5, 50and 57° C.), see Table 19.

EDTA (Ethylene Diamine Tetraacetic Acid) (5 mM) as a chelating agent atpH 4.5 and 50° C. did not change CMCase activity. At pH 4.5 (57° C.) andat pH 7.0 (50° C.) EDTA caused slight decreases in CMCase activity. AtpH 7.0 and 57° C., EDTA caused slight increase in CMCase activity, seeTable 19.

Non-ionic detergent Tween-80 (3 g/L, polyoxyethylene sorbitanemonooleate), did not change CMCase activity of C1 (at pH-s 4.5 and 7.0and at 50 and 57° C., see Table 19.

Oxidizing agent persulfate (3 g/L) did not change CMCase activity of C1(at pH-s 4.5 and 7.0 and at 50 and 57° C.), see Table 19.

C1 CMCase is resistant to serine proteases (trypsin and chymotrypsin),chelating agents (EDTA, citrate), non-ionic detergent (Tween-80) and tooxidizing agent (persulfate).

TABLE 19 The effect of citrate, EDTA, Tween-80 and persulfate onactivity of C1 cellulasese (# 47.9.1). Incubation time - 3 hours.Temperature CMCase activity Effector Concentration (° C.) pH remaining(%) None (control) — 50 4.5 100 Citrate 0.1M 50 4.5 100 EDTA 5 mM 50 4.5100 Tween-80 3 g/L 50 4.5 100 Persulfate 3 g/L 50 4.5 97 None (control)— 57 4.5 62 Citrate 0.1M 57 4.5 65 EDTA 5 mM 57 4.5 60 Tween-80 3 g/L 574.5 68 Persulfate 3 g/L 57 4.5 65 None (control) — 50 7.0 78 EDTA 5 mM50 7.0 50 Tween-80 3 g/L 50 7.0 52 Persulfate 3 g/L 50 7.0 50 None(control) — 57 7.0 30 EDTA 5 mM 57 7.0 38 Tween-80 3 g/L 57 7.0 25Persulfate 3 g/L 57 7.0 30

EXAMPLE 11 Stone Wash Tests of Cellulase Samples Produced by DifferentStrains of Chrysosporium

Preparations #-s 47.1000, 47.1001, 47.2000 and 47.2001 produced bydifferent strains of Chrysosporium were used for wash test with 2-Lspecial wash machine at pH 6.5, 50° C., during 60 min with 135 g ofdesized denim jean fabric. Total amount of CMCase activity per trial wasconstant and equal to 336 U/run. After drying abrasion and backstainingof garment was evaluated by Datacolor measurement. The results arepresented in Table 20. The results show that cellulases produced fromdifferent strains of Chrysosporium demonstrate similar wash performanceat neutral pH in terms of abrasion and backstaining levels to thecellulases produced by the C-1 species Chrysosporium lucknowense Garg1966.

TABLE 20 Stone wash activity of cellulase preparations from differentstrains of Chrysosporium Preparation # Abrasion (*) Backstaining (**)47.1000 12.3 1.6 47.1001 12.1 1.6 47.2000 14.2 1.7 47.2001 14.6 1.6 (*)reflectance from front side, the higher value the more abrasion, blank =9.1 (**) reflectance from back side, the higher value the morebackstaining

EXAMPLE 12 Purification of Cellulase Components

1. Selection of the C1 samples for purification. The C1 cellulasepreparation #47.11.1 was chosen for further purification in view of thefact that 47.11.1 possessed (i) high protein content; (ii) high FPA andCMCase activity (see Table 4).

2. Isolation and purification of C1 complex component. The firstpurification step included ion exchange chromatography on aDEAE-Toyopearl column (TosoHaas, Japan). Dry C1 cellulose preparation(1.5 g) was dissolved in 15 mL of 0.01 M Na-phosphate buffer, pH 7. Thesolution was centrifuged and the supernatant desalted using an AcrylexP-2 column. The desalted sample was then applied to the DEAE-Toyopearlcolumn (1.5×30 cm) in 0.03 M phosphate buffer, pH 4.7 and adsorbedproteins were eluted in 0-0.2 M NaCl gradient with flow rate of 1mL/min. Three pooled fractions were obtained—the non-bound (NB) fractionwas eluted in the start buffer, Fractions I and II were eluted across a0-0.2 M NaCl gradient. All fractions possessed cellulolytic activities.

3. SDS-PAGE of protein fractions. After sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the NB Fraction includedproteins with molecular weights from 30 and 70 kD. Fraction I includesproteins with molecular weights from 25 to 100 kD and Fraction IIcontained proteins with molecular weights from 35 and 100 kD. SDS-PAGEwas carried out with a 10% separating gel (100×80×0.75 mm) underdenaturing conditions. Reagents and kits were obtained from Bio-Rad(USA). Coomassie brilliant blue R-250 in 25% trichloracetic acid wasused for protein staining.

4. IEF of Protein Fractions. After isoelectric focusing (IEF), the NBFraction includes proteins with isoelectric points (pI's) of from 4.6 to8.0. Fraction I contains proteins with pI values from 3.2 to 5.5, andFraction II conatins proteins with pI from 3.0 to 5.5. Isoelecticfocusing was carried out with 7% PAAG in mini-IEF Model 111 (fromBio-Rad). Reagents and kits were obtained from Bio-Rad (USA). Coomassiebrilliant blue R-250 in 25% trichloroacetic acid was used for proteinstaining.

5. pH-dependencies of CMCase activity of protein fractions. Table 21represents the pH dependencies of CMCase and RBB-CMCase activities ofFractions NB, I and II of C1 cellulase. The following buffer systemswere used: acetate buffer (pH 4-5), phosphate buffer (pH 6-8), andcarbonate buffer (pH 8.5-10). In addition, a universal buffer system wasused which consisted of acetate, borate, and phosphate (pH 4-10).

TABLE 21 The effect of pH on CMCase and RBB-CMCase activities of C1protein fractions pH NB Fraction Fraction I Fraction II CMCase activity,50° C. (%) 4.0 85 70 95 4.5 95 85 100 5.0 100 90 95 5.5 90 100 90 6.0 8090 80 6.5 70 85 80 7.0 65 85 80 7.5 60 65 75 8.0 50 60 60 8.5 45 50 509.0 30 45 40 9.5 10 40 32 RBB-CMCase activity, 50° C. (%) 4.5 65 95 1005.0 100 100 95 5.5 95 100 90 6.0 95 95 90 6.5 80 87 90 7.0 80 85 87 7.560 65 85 8.0 50 60 70 8.5 45 60 60 9.0 30 50 40 9.5 10 40 32

CMCase and RBB-CMCase activities of the NB, I and II Fractions followingDEAE-Toyopearl ion exchange chromatography had a non-symmetric bell-typepH profile. CMCase activity of the NB Fraction showed a maximum at pH4.5-5.5 and 50% of maximal activity at pH 8.0. RBB-CMCase activity ofthe NB Fraction had a maximum at pH 5.0-6.0 and 50% of maximal activityat pH 8.0. CMCase activity of Fraction I had a maximum at pH 5.0-6.0 and50% of maximal activity at pH 8.5. RBB-CMCase activity of Fraction I hada maximum at pH 4.5-7.0 and 50% of maximal activity at pH 8.5. CMCaseactivity of Fraction II had a maximum at pH 4.0-5.5 and 50% of maximalactivity at pH 8.5. RBB-CMCase activity of Fraction II had a maximum atpH 4.5-7.5 and 50% of maximal activity at about pH 8.5.

6. Stability of CMCase of protein Fraction I (after DEAE-Toyopearl).Table 22 shows temporal CMCase activity curves of Fraction I afterDEAE-Toyopearl ion exchange chromatography at different pH (5.2-8.7) and50° C. CMCase activity of Fraction I was most stable at pH 5.2-7.2(between about 30% and 45% of activity was lost over 3 hours). At pH7.7, 60% of activity was lost after about 1 hour, whereas at pH 8.3 and8.7, 50% of activity was lost after about 0.5 hour. At pH 8.3, 100% ofCMCase activity was lost after 3 hours, and at pH 8.7, 100% of activitywas lost after 2 hours.

TABLE 22 Stability of CMCase of a protein fraction I afterDEAE-Toyopearl at 50° C. CMCase activity remained (%) Time (h) pH 5.2 pH7.2 pH 7.7 pH 8.3 pH 8.7 0 100 100 100 100 100 0.5 97 75 65 50 50 1 9055 40 25 15 2 40 50 35 10 0 3 30 45 25 0 0

7. Properties of Fractions after Ion Exchange Chromatography onDEAE-Toyopearl. Adsorption experiments using Avicel (micro crystallinecellulose) as a substrate demonstrated that fractions I and II did notbind to a crystalline substrate and the NB Fraction bound to Avicel witha distribution coefficient of 0.2 L/g. Specific activities of the NB, Iand II Fractions toward different substrates are presented in Table 23.All three fractions possessed CMCase, endoglucanase, avicelase,β-glucanase and xylanase activities, but the NB Fraction had noβ-glucosidase activity, contrary to Fractions I and II. The Micro DenimWash test for Fractions NB, I and II showed that Fractions I and IIpossessed approximately equal activity on denim at pH 7 while the NBFraction showed lower activity (according to the Micro Denim Wash test).

TABLE 23 Specific Activities of Fractions After Ion ExchangeChromatography on DEAE-Toyopearl Specific Activities, U/mg Protein MicroDenim Fractions CMCase Endoglucanase Avicelase β-glucosidase β-glucanaseXylanase Wash Test NB 9.9 6.2 0.18 0.0 11.0 5.3 + I 3.7 2.3 0.02 3.6 1.90.7 ++ II 0.8 0.2 0.02 0.02 1.2 0.04 ++

8. Micro Denim Wash Test. This test was carried out using 20 mL ofbuffered enzyme solution having a preset level of CMCase activity. RealIndigo-denim stained swatches were treated at 50° C. for 60 minutes inthe conditions of excessive mechanical stress (abrasion). The level ofcellulase performance was evaluated by panel score according to thecolor reduction after the samples were dried. Color measuringinstruments and software could also be used. More “+” values indicatebetter abrasion.

9. Further Purification of Proteins from Fraction I After Ion ExchangeChromatography on DEAE-Toyopearl. Desalted Fraction I (25 mL, 0.8 g/l ofprotein) was subjected to Macro Prep Q ion exchange chromatography. TheMacroPrep Q column (1.5×10 cm) was equilibrated with 0.03 M acetatebuffer, pH 4.7, and the adsorbed proteins eluted in 0-0.1 M NaClconcentration gradient. Three fractions, I.1, I.2, and I.3, werecollected. Fraction I.2 showed the highest Micro Denim Washing activityat pH 7 and fraction 1.3 showed the lowest. SDS-PAGE results showed thatFractions I.1 and I.2 differed by the presence of a low molecular weightprotein (25 kD) in Fraction I.2, which might have accounted for thestonewashing activity. Fraction I.1 had a pI of 4.2 as shown by IEFmeasurements. Fractions I.1 and I.3 had too low a protein content topermit further study. Therefore, Fraction I.2 was used in furtherpurification.

The next purification step involved chromatofocusing on a Mono P column.Fraction I.2 was equilibrated with 0.03 M imidazole buffer, pH 6.8, andapplied to the column. Adsorbed proteins were eluted with Polybuffer 74(1:8), pH 4.0, whereupon 2 major peaks in protein and activity profileswere observed. The first peak, designated Peak A, showed lower specificCMCase activity (2.5 Units/mg) compared to Peak B (3.6 Units/mg). Nativepolyacrylamide gel electrophoresis showed the presence of 2 proteinbands in Peak A, with the higher molecular weight protein band beingactive toward CMC stained with Congo Red. One active protein band undernative conditions was observed in Peak B. SDS-PAGE data showed that PeakA included 2 major proteins (60 kD and 70 kD) and Peak B contained onemajor protein (25 kD) and one minor protein (27 kD). The fractioncollected within Peak B was designated as 25 kD-endoC1 and was used infurther studies. Table 24 shows the specific activities of 25 kD-endoC1toward different substrates. The 25 kD-endoC1 had CMCase, RBB-CMCase,endo-glucanase, FPA, avicelase, β-glucanase, and xylanase activities butdid not show β-glucosidase activity. This combination of differentactivities shows that 25 kD-endoC1 is an endoglucanase. The pH optimumof CMCase and RBB-CMCase activities is approximately 6.0 (Table 24). The25 kD-endoC1 possessed high stonewashing activity (according to a MicroDenim Wash test using cotton swatches) (see Table 24).

During elution of Peak A from the Mono P column a number of fractionswere recovered that differed in the ratio of 60 kD to 70 kD proteins,especially a fraction designated 70(60) kD-C1 that includedpredominantly a 60 kD protein and a fraction designated 70 kD-endoC1with predominantly a 70 kD protein. Specific activities of thesefractions toward different substrates are presented in Table 24. As isseen therein, the 70(60) kD-C1 fraction possessed low specificendoglucanase (0.5 U/mg) and a high specific avicelase (0.31 U/mg)activities compared to the 70 kD-endoC1 fraction (2.8 U/mg endoglucanaseand 0.18 U/mg avicelase) and had very low β-glucosidase activity.Specific activities toward FP, β-glucan, and xylan of the 70(60) kD-C1fraction were low (see Table 24) and only cellobiose was formed as aproduct of avicel hydrolysis. Stonewashing activity (according to aMicro Denim Wash test using cotton swatches) of the 70(60) kD-C1fraction was low (see Table 24). These data show that the 60 kD proteinfrom Fraction I after ion exchange chromatography on DEAE-Toyopearl canbe designated as a cellobiohydrolase. The pH optimum of 70(60) kD-C1(toward CMC and RBB-CMC) was approximately 5.0 (Table 24).

The 70hkD-endoC1 had high specific CMCase, RBB-CMCase, endoglucanase,FPA, β-glucanase and xylanase activities and possessed some avicelaseand β-glucosidase activity (Table 24). The 70 kD-endoC1 also possessedrelatively high stonewashing activity (according to the Micro Denim Washtest using cotton swatches) (see Table 24). The 70 kD-endoC1 fromfraction I after ion exchange chromatography on DEAE-Toyopearl appearsto be an endoglucanase. As seen from Table 24, the pH optimum for 70kD-endoC1 is approximately 6.0 for both the CMCase and RBB-CMCaseactivities.

10. Further Purification of Proteins from Fraction II after Ion ExchangeChromatography on DEAE-Toyopearl. Fraction II, obtained as a result ofDEAE chromatography, was divided into 3 fractions (Fraction II.1, II.2,and II.3, respectively) using a longer 0-0.2 M NaCl gradient (over aperiod of 8 hr) on a DEAE-Toyopearl column. Results of SDS-PAGE showedthat Fraction II.1 included 2 major proteins of molecular weight 60 kDand 100 kD, Fraction II.2 included 3 major proteins of 35 kD, 60 kD, and100 kD, and Fraction II.3 included 2 major proteins of 43 kD and 60 kD.Fraction II.3 demonstrated the highest CMCase activity (10 units/mg ofprotein) but showed low washing activity (using a sub-Micro Denim Washtest) and specific CMCase activity of 1 unit/mg. Fraction II.2 did notshow any of the washing activity (but CMCase activity was 0.7 U/mg).Thus, Fractions II.1 and II.3 were purified further.

TABLE 24 Properties of C1 Enzymes 60 kD 43 kD 60 kD 25 kD 70 (60) 70 kDendo 100 kD endo endo pH endo kD endo (II.1) (II.1) (II.3) (II.3) CMCaseActivity (U/mg) 5.0 4.6 1.10 3.5 0.70 0.03 1.02 1.32 6.0 5.0 0.83 3.80.52 0 1.01 1.07 7.0 3.9 0.65 3.0 0.45 0 0.90 1.03 RBB-CMCase (U/mg) 5.08.2 0.12 1.5 0.90 0 0.82 1.21 6.0 8.8 0.10 2.7 0.84 0 0.75 1.14 7.0 6.60.07 2.4 0.68 0 0.73 1.12 FPA (U/mg) 5.0 1.0 0.17 0.61 0.31 0 0.45 0.52Endoglucanase (viscometric) (U/mg) 5.0 2.26 0.50 2.8 0.21 0 0.15 0.27Avicelase (U/mg) 5.0 0.16 0.31 0.18 0.03 0 0.01 0.01 β-Glucosidase(U/mg) 5.0 0 0.02 0.16 0.02 0 0.02 0 β-Glucanase (U/mg) 5.0 0.66 0.072.4 1.3 0 3.9 4.4 Xylanase (U/mg) 5.0 0.40 0.16 0.50 0.06 0.01 0.07 0.01Micro Denim Wash Activity 5.0 +++ − ++ n.d. n.d. n.d. n.d. 7.0 +++ − ++n.d. n.d. n.d. n.d. Sub-Micro Denim Wash Activity 5.0 n.d. n.d. n.d. +++− + + 7.0 n.d. n.d. n.d. +++ − +++ ++ n.d. = not determined

11. Sub-Micro Denim Wash Test. This test was performed on fragments ofreal Indigo-stained denim in 2 mL of buffered enzyme solution (2 unitsCMCase activity) at 50° C. for 2 hours in the conditions of excessivemechanical stress. The level of cellulase performance was evaluated bypanel score according to the color reduction after the samples weredried.

12. Purification of Fraction II.1. Fraction II.1 was applied to a Macroprep Q column equilibrated with 0.03 M acetate buffer, pH 4.75, and theadsorbed proteins were eluted in a NaCl gradient (0-0.3 M). Two proteinpeaks were obtained but only the first one showed CMCase activity.SDS-PAGE of the material from the first peak showed that proteins, with60 kD and 100 kD were isolated in a homogeneous state. According to IEFdata, the 60 kD and 100 kD proteins possessed an acidic pI of about 3.The activities of the 60 kD and 100 kD proteins toward differentsubstrates are shown in Table 24. The 60 kD protein designated as 60kD(II.1)-endoC1 was found to possess endoglucanase, CMCase, RBB-CMCase,FPA and β-glucanase activities at pH 5 (0.2, 0.7, 0.9, 0.3, and 1.3units/mg of protein, respectively, as shown in Table 24). Avicelase,β-glucosidase, and xylanase activities were rather low. This combinationof activities shows that the 60 kD(II.1)-endoC1 is an endoglucanase. The60 kD(II.1)-endoC1 also possesses high washing activity (by theSub-Micro Denim Wash test) both at pH 5 and at pH 7 (per Table 24). ThepH dependence of CMCase and RBB-CMCase activities for this proteinshowed maxima at pH 4.0-4.5, with 50% of maximal activity toward CMC and85% of maximal activity toward RBB-CMC retained at pH 6, and 15-20% ofboth activities retained at pH 9 and 10 (see Table 25).

The 100 kD protein from Fraction II.1 was designated as 100kD(II.1)protein and almost did not have cellulase activity (Table 24). Thisprotein possessed only very low CMCase (0.03 U/mg) and xylanase (0.008U/mg) activities and could not be determined to be a cellulytic enzyme.According to the Sub-Micro Denim Wash test, the 100 kD (II.1) proteindid not demonstrate any stonewashing activity (Table 24) and also failedto show any protease activity at either pH 5 or pH 7.

13. Purification of Fraction II.3. Fraction II.3 was also purified byMacro Prep Q chromatography. The adsorbed proteins were eluted in0.2-0.6 M NaCl gradient (the start buffer was 0.03 M acetate, pH 4.75).SDS-PAGE of the obtained fractions after the Macro Prep Q chromatographyshowed that the 43 kD and 60 kD proteins were obtained in homogeneousform. Isoelectrofocusing of these fractions showed that both the 43 kDand 60 kD proteins had pI values of about 3. The 43 kD and 60 kDproteins were designated 43 kD(II.3)-endoC1 and 60 kD(II.3)-endoC1,respectively. The activities of these enzymes toward differentsubstrates (see Table 24) showed that they had similar specific CMCase,FPA, avicelase, and xylanase activities. The 60 kD(II.3)-endoC1possessed higher specific RBB-CMCase, endoglucanase and FPA activities(Table 24). At the same time it should be stressed that the 43kD(II.3)-endoC1 and 60 kD(II.3)-endoC1 possessed very littlestonewashing activities at pH 5 (using the Sub-Micro Denim Wash test).However, both 43 kD(II.3)-endoC1 and 60 kD(II.3)-endoC1 demonstratedremarkable stonewashing activity at pH 7, and at the same time the 43kD(11.3)-endoC1 had higher stonewashing activity compared to the 60kD(II.3)-endoC1. As seen from the pH dependencies in Table 25, 43kD(II.3)-endoC1 showed a broad pH optimum (from pH 4.5 to 8) in the caseof both CMCase and RBB-CMCase activities. The 43 kD(II.3)-endoC1possessed 50% CMCase and 70% RBB-CMCase activities from a maximum at pH9 and 20% of both CMCase and RBB-CMCase at pH 10. In contrast, 60kD(II.3)-endoC1 had a narrow pH optimum at pH 4-4.5 toward CMC and abroad pH optimum (from pH 4 to 8) toward RBB-CMC and 30% RBB-CMCaseactivity being retained at pH 9.

It should be noted that in all cases of purified proteins disclosedherein, molecular weights were determined using gel electrophoresis(especially SDS-PAGE) and reference proteins of known molecular weightas standards. As with all analyses using such methods, the results areonly approximate and some variation in molecular weight may be observedas different gels are run by different workers using different sets ofmolecular weight standards as references.

Such purified, and partially purified, enzyme preparations are highlyuseful as components of detergent, fabric softening, depilling, colorbrightening and stone washing compositions. Thus, the above isolated andpurified enzyme preparations find utility in such applications accordingto the present invention. Thus, methods for stone washing, fabricsoftening, depilling, color brightening and cleansing as heretoforerecited herein, as well as typical methods for accomplishing suchapplications as already disclosed in the literature will readily employsuch purified, or partially purified, enzyme preparations, andcompositions containing such, as main or additive agents in effectingthe goals of such procedures. The use of other and different purified,or partially purified, enzyme preparations in such applications is knownin the literature with many enzymes in commercial use.

TABLE 25 The Effect of pH on CMCase and RBB-CMCase activities of C1Cellulase Enzymes pH 60 kD(II.1)-endo 43 kD(II.3)-endo 60 kD(II.3)-endoCMCase Activity, 50° C. (%) 3.5 85 80 85 4.0 100 100 100 5.0 65 100 856.0 50 100 70 7.0 45 90 65 8.0 25 70 45 9.0 17 30 30 10.0 15 20 10 11.015 5 5 RBB-CMCase activity, 40° C. (%) 3.5 85 85 80 4.0 100 100 100 5.090 100 100 6.0 85 95 95 7.0 70 90 90 8.0 50 80 85 9.0 20 70 70 10.0 1520 30 11.0 15 15 15

It should be understood that the neutral and/or alkaline cellulasesheretofore described can have different enzymatic activities dependingon the chemical structure of the substrate used in measuring theactivity and the particular assay method employed to measure activity.Thus, the purified, or partially purified, neutral and/or alkalinecellulases will show different pH/activity profiles depending on theassay method and substrates employed. To resolve any confusion as to thenature of the activities and properties of the substantially purifiedcellulase enzymes prepared by the methods of this example, the followingis a description of the activities and properties measured for thecellulases of the purified fractions.

The purified, or partially purified, cellulases prepared herein showedboth endoglucanase and/or cellobiohydrolase activity when theappropriate substrate was employed. Thus, the cellulase preparationsthat showed endoglucanase activities all had pI values between about 3and about 4.5. More specifically, these fractions included cellulases(endoglucanases) having molecular weights and pI values as follows: MWabout 25 kD (pI about 4.0), MW about 70 kD (pI about 4.2), MW about 60kD (pI about 3.0) and MW about 43 kD (pI about 3.1). These cellulasefractions also contained proteins showing a cellobiohydrolase activity.More specifically, the latter had a MW of about 60 kD and pI about 4.2.

Methods of using the compositions and purified enzymes according to thepresent invention have been well disclosed in the literature, includingmany patents, whose disclosures are hereby incorporated by reference.These would include Clarkson (U.S. Pat. No. 5,290,474), which disclosesuse of cellulase enzymes and cellulase enzyme-containing compositions,including surfactants and other additives, for use in aqueous washmedia, detergent compositions, media designed to enhance color retentionand/or restoration, as well as imparting improved softening and feelproperties, especially to cotton-containing fabrics. The cellulaseenzymes and cellulase-containing compositions according to the presentinvention are also intended for use in the same applications, specificdescriptions of which are described in many, if not all, of thereferences cited. In addition, the utility of cellulase enzymes andcompositions for applications such as harshness reduction, or fabricsoftening, is also taught in Barbesgaard et al (U.S. Pat. No.4,435,307), specifically disclosing the use of fungal cellulases, butnot those of the genus Chrysosporium, at alkaline pH ranges andincluding various additive agents, such as those employed in conjunctionwith the novel cellulase and cellulase compositions of the presentinvention, for harshness reduction, or fabric softening, and washing asa single operation. The cellulases and cellulase compositions of thepresent invention are similarly useful and the teachings of Barbesgaardwith respect to such applications is specifically incorporated herein.In addition, the use of cellulase enzymes and cellulase compositions,other than the novel cellulases and cellulase compositions of thepresent invention, for applications to color brightening arespecifically disclosed in Boegh (European Patent EP 0 220 016), whichteaching is specifically incorporated herein.

Of course, the novel cellulase enzymes and cellulase compositions of thepresent invention will be understood by those of skill in the art to behighly useful for the same applications as disclosed in in the foregoingreferences, thus rendering the elucidation of any further details ofsuch applications unnecessary. However, such purified, or partiallypurified, enzymes and enzyme-containing compositions are also useful insuch applications as deinking and biobleaching of paper or pulpmaterials and method of doing so will readily suggest themselves tothose of skill in the art, especially after they review the teachingsherein.

EXAMPLE 13 C-1 Cellulase Production in 60 Liter Batch Fermentor

1. Inoculum Preparation

Inoculum preparations or starter cultures for the batch fermentationwere prepared as follows. One milliliter (1 ml) of C-1 spore culture wasused to inoculate each of two flasks to generate a total of 2.0 litersof inoculum. The starter culture was incubated at 150 rpm, at 30° C. for56 hours.

Medium for Inoculum Preparation* K₂HPO₄ 0.5 g/L MgSO₄•7H₂O 0.15 KCl 0.05FeSO₄•7H₂O 0.007 yeast extract (ohly KAT) 1.0 peptone (Hormel PSR 5)10.0 lactose 10.0 glucose 10.0 *The pH of the inoculum medium wasadjusted to pH 7.0 with NaOH, the media was then autoclaved for 35minutes at 121° C. in two six liter baffled flasks each containing oneliter of medium.

2. Cellulase Production in 60 Liter Batch Fermenter (Preparation of47.0325)

The two liter shaker flask inoculum culture prepared above, was usedinoculate 40 liters of medium contained in a 60 liter fermenter. Themedium for fermentation was as follows:

Fermentation Medium* K₂HPO₄ 0.22 g/L KH₂PO₄ 0.08 g/L (NH₄)₂SO₄ 4.0 g/LNa₃citrate•2H₂O 4.0 g/L MgSO₄•7H₂O 0.03 g/L CaCl₂•2H₂O 0.4 g/LFeSO₄•7H₂O 0.5 mg/L MnSO₄•7H₂O 0.5 mg/L ZnSO₄•7H₂O 0.2 mg/L CoCl₂•6H₂O0.24 mg/L lactose 5.0 g/L yeast extract (ohly KAT) 0.05 g/L defattedcotton seed flour 5.0 g/L (Pharmamedia) cellulose (Signmacell 50) 20.0g/L pH 7.0 *The 40 liters medium was in deionized water, and wassterilized for 45 minutes at 121° C.

-   -   After inoculation of the fermentation medium, pH was maintained        above 6.9 by addition of NH₃ and below 7.1 by addition of H₂S0₄.        The fermenter was incubated for 64 hours with agitation and        aeration as necessary to maintain dissolved oxygen greater than        30% of saturation.

3. Recovery of Cellulase Activity

Suspended solids from the fermented culture were removed by filtrationon large Buchner funnel using Whatman 54 filter paper and 10 g/L Celite503 as filter aid. The filtrate was collected, and the cellulaseconcentrated by ultrafiltration using 10,000 MW cutoff hollow fiberfilter. The concentrate was freeze dried. The dried concentrate wasdesignated cellulase preparation 47.0325. The activity of thispreperation is given in Table 4.

EXAMPLE 14 Mutation Procedure Used to Generate Mutant Stain of C-1

A spore suspension was prepared using a Pridham agar plate (4 g/L yeastextract, 10 g/L malt extract, 10 g/L glucose, 15 g/L agar) containing asporulated culture of strain C1. The plate was flooded with 10 ml of0.05% Tween 80. The suspension was transferred to a sterile screw captube and vortexed on high for 1 minute. The suspension was then filteredthrough a column to remove mycelium. Spores were counted and diluted to7×10⁵ spores per ml in water. Ten mls of the spore suspension weretransferred to a standard glass petri dish. The spores were irradiatedfor 75 seconds at 720 μWatts/cm² using a Pen-Ray UV bulb. The sporesuspension was gently stirred throughout the irradiation using a sterilepaper clip as a magnetic stir bar. Following irradiation, the sporesuspension was taken to a foil wrapped tube, diluted in water and platedin dim light to NH₄ minimal medium as defined below. After incubating 20days at 30 degrees C., a colony was identified as a large colony with alarge zone of cellulose clearing around the colony.

NH₄ Minimal Medium, pH 7.5

-   1 g/L K₂HPO4₄-   0.1 g/L KCl-   0.3 g/L MgSO₄.7H₂O-   0.1 g/L NaCl-   16 mg/L FeCl₃.6H₂O-   1.92 g/L(NH₄)₂SO₄-   15 g/L Difco Noble agar-   2.5 g/L acid swollen cellulose (added as a 1.25% stock after    autoclaving)-   0.5 g/L sodium deoxycholate (added after autoclaving)

EXAMPLE 15 C-1 Mutant Cellulase Production in 60 Liter BatchFermentation Flasks (Preparation of 47.0528)

1. Inoculum Preparation

Preparations of starter cultures for the fed batch fermentation wereprepared as described in Example 13 (section 1).

2. Cellulase Production In 60 Liter Batch Fermentation

The two liter inoculum was used to inoculate 40 liters of fermentationmedium as described below.

Fermentation Medium* K₂HPO₄ 0.44 g/L KH₂PO₄ 0.16 g/L (NH₄)₂SO₄ 3.0 g/LNa₂citrate•2H₂O 4.0 g/L MgSO₄•7H₂O 0.06 g/L CaCl₂•2H₂O 0.8 g/LFeSO₄•7H₂O 0.1 mg/L MnSO₄•7H₂O 0.04 mg/L ZnSO₄•7H₂O 0.04 mg/L CoSO₄•6H₂O0.048 mg/L lactose 5.0 g/L yeast extract (ohly KAT) 0.1 g/L defattedcotton seed flour 10.0 g/L (Pharmamedia) cellulose (Sigmacell 50) 20.0g/L *The 40 liters medium was in deionized water, and sterilized byautoclaving for 45 minutes at 121° C.

3. Fermentation Conditions

The pH was maintained at around 7.0 and controlled by addition of NH₃ atpH above 6.9, and addition of H₂SO₄ at pH below 7.1. Incubation time was87 hours, agitation and aeration were as necessary to maintain dissolvedoxygen greater than 30% of saturation. At 40 hours, 3.0 liters of feedsolution as described below, was added at a rate of 5.0 ml each 5minutes.

Feed Solution for Fermenter K₂HPO₄ 0.88 g/L KH₂PO₄ 0.32 g/L (NH₄)₂SO₄4.0 g/L Na₂citrate•2H₂O 4.0 g/L MgSO₄•7H₂O 0.12 g/L CaCl₂•2H₂O 0.16 g/LFeSO₄•7H₂O 0.2 mg/L MnSO₄•7H₂O 0.08 mg/L ZnSO₄•7H₂O 0.08 mg/L CoCl₂•6H₂O0.096 mg/L lactose 20.0 g/L yeast extract (ohly KAT) 0.2 g/L Pharmamedia20.0 g/L cellulose (Sigmacell 50) 20.0 g/L

4. Recovery of Cellulase Activity

Suspended solids were removed by filtration on large Buchner funnelusing Whatman 54 filter paper and 10 g/L Celite 503 as filter aid. Thefiltrate was collected and cellulase concentrated by ultrafiltrationusing 10,000 MW cutoff hollow fiber filter. The concentrate was dried byfreeze-drying. The concentrate was designated cellulase preparation47.0528 (activity is given in Table 4).

EXAMPLE 16 Assay of Cellulase Activity Using Cellazyme Assay

This assay was carried out using Cellazyme C tablets and the assay kitobtained from Megazyme (Aust) Pty. Ltd., Sydney, NSW 2101, Australia.The substrate used is Azurine-crosslinked HE-celulose (AZCL-Cellulose)supplied commercially as cellazyme C tablets, ready for use. Briefly,0.5 mL aliquots of enzyme preparation (diluted if necessary) in 0.025 Macetate buffer (pH 4.5) are equilibrated to 40° C. for 5 minutes inglass test tubes (16×122 mm). The test reaction is then initiated byaddition of a Cellazyme C tablet (without stirring). After exactly 10minutes at 40° C., the reaction is terminated by addition of Trizma Basesolution (10.0 mL, 2% w/v, from Sigma Chemical Co., St. Louis, Mo.) andthen vortexing. The tubes are then allowed to stand for about 5 minutesat room temperature whereupon the slurry is stirred again, filteredthrough a Whatman No. 1 (9 cm) filter circle and absorbance of thefiltrate measured at 590 nm. The absorbance measurements are readagainst a blank containing both substrate and rt. 15 enzyme but preparedby adding the Trizma Base to the enzyme solution prior to addition ofthe cellazyme C tablet. This slurry is left at room temperature ratherthan 40° C. A single blank is used for each set of determinations andwas used to zero the spectrophotometer.

In this assay, one unit of enzyme activity is defined as the amount ofenzyme required to release one micromole of glucose reducing sugarequivalents per minute under the defined assay conditions.Endo-cellulase activity was then determined by reference to a standardcurve (a sample curve was supplied with the kit but could be readilygenerated in the laboratory, if different particular enzyme, enzymedilution and conditions are to be employed for a given set ofexperiments).

Using this assay for our own neutral/alkaline enzyme activity, we foundendo-Cellulase activity (taking the activity at pH 7 as 100%) to be92.4% (at pH 6), 75.6% (at pH 5.0) and 69.7% (at pH 4.0).

EXAMPLE 17 Detergent Wash Test for De-Pilling and Color Brightening(Anti-Fading)

Tests were carried out according to the AATCC monograph,“Standardization of Home Laundry Test Conditions” in the AATCC TechnicalManual, 1997 (revised 1995), using a regular Kenmore home top-loadingwasher, using a Kenmore home tumble-dryer. Three different styles of redadult-size socks (88% cotton, 10% polyester, 2% lycra) were used as testgarments (plain ribbed knit, heavy ribbed knit and waffle weave). Thesocks were divided into 3 groups with an equal number of each style ineach group, with a non-washed garment of each style used as a reference.The approximate weight of each group was 1 kg.

The samples of detergent were prepared prior to each wash as follows: a)Cheer Triple Guard (as purchased in the U.S.A. and commonly at alkalinepH) as is (to demonstrate the de-pilling and color care properties ofcellulases originally present in Cheer, b) thermally treating the sampleof Cheer to inactivate all of the enzymes originally present in thedetergent (to limit the performance of Cheer to its components otherthan enzymes. For thermal inactivation, a 40 g sample of Cheer wassuspended in 200 mL of water and heated on high (1000 W) in a homemicrowave for 5 minutes, the final temperature reached being 95° C.Then, repeating the same steps (a) and (b) above, but adding 5 g of C1to the preparation.

The wash/dry cycles were performed 25 times for each group. Each groupwas washed throughout the 25 wash cycles using 1 kg of garments to 20liters of wash liquor (water level in the washer was low) and 40 g ofonly one of the above mentioned detergent preparations. The temperaturewas set on hot-hot, the wash duration was 30 minutes, followed byregular, high speed centrifugation. The dryer temperature was set onhigh and the drying cycle lasted 45 minutes. The testing was completeafter 25 washes, when the garments were evaluated, by several differentgroups/panels skilled in the art, for de-pilling and color brightening.

A summary of the procedure is as follows:

Inactivation of original Addition of Enzymes Preparation(s) Cheerenzymes from AARL a. Cheer (complete) No No b. Cheer (no enzymes) Yes Noc. Cheer + C1 Yes Yes (5 g of C1)

The results of the rating for each wash group were as follows (where all3 styles of socks were rated the same):

Cheer Cheer (complete) (enzymes inactivated) Cheer + C1 De-pilling ++++− +++ Color Brightening ++ − ++++

It should be understood that the examples and embodiments describedherein are intended for illustrative purposes only and that variousmodifications or changes in light thereof will readily suggestthemselves to persons skilled in the art and are to be included withinthe spirit and purview of this application and the scope of the appendedclaims.

1. A composition having neutral and/or alkaline cellulase activity,obtained by a method which comprises growing a wild type or mutantfungus of the genus Chrysosporium in culture in a suitable medium,wherein the fungus is Chrysosporium lucknowense, Chrysosporium pannorum,Chrysosporium keratinophilum, Chrysosporium lobatum, Chrysosporiummerdarium, Chrysosporium queenslandicum, or Chrysosporium tropicum.
 2. Acomposition according to claim 1 having optimal cellulose activity at atemperature from about 40° C. to about 60° C., at a pH from about 5.0 toabout 12.0.
 3. A composition according to claim 1 having at least 50% ofthe optimal cellulase activity, at a pH from about 6.0 to about 7.0, ata temperature from about 40° C. to about 60° C.
 4. A compositionaccording to claim 1 wherein said cellulase activity is assayed by anyone of the CMCase, RBBCMCase, endoviscometric or filter paper activityassays.
 5. A substantially purified and isolated protein fraction,obtained from a composition according to claim 1, and having at least50% of its maximal cellulase activity at a pH between about 6.0 andabout 7.0 as measured by any one of the CMCase, RBBCMCase,endoviscometric or filter paper activity assays.
 6. An endoglucanaseobtained from a fraction according to claim 5, having a molecular weightof about 25 kD and pI of about
 4. 7. An endoglucanase obtained from afraction according to claim 5, having a molecular weight of about 70 kDand a pI of about
 4. 8. An endoglucanase obtained from a fractionaccording to claim 5, having, a molecular weight of about 60 kD and a pIof about
 3. 9. An endoglucanase obtained from a fraction according toclaim 5, having a molecular weight of about 43 kD and a pI of about 3.10. A cellobiohydrolase obtained from a fraction according to claim 5,having a molecular weight of about 60 kD and a pI of about
 4. 11. Asubstantially purified and isolated neutral and/or alkaline cellulaseenzyme, isolated from a protein fraction according to claim 5, andhaving a pI of between about 3 and about 5.5.
 12. A cellulase accordingto claim 11 wherein said cellulase possesses either endoglucanase orcellobiohydrolase activity.
 13. A cellulase according to claim 12wherein said cellulase retains at least 50% of its maximal cellulaseactivity at a pH between about 6.0 and about 7.0.
 14. An endoglucanaseobtained from a fraction according to claim 5 and having a molecularweight of about 25 kD.
 15. An endoglucanase obtained from a fractionaccording to claim 5 and having a molecular weight of about 70 kD. 16.An endoglucanase obtained from a fraction according to claim 5 andhaving a molecular weight of about 43 kD.
 17. A detergent compositioncontaining one or more purified enzymes isolated from a protein fractionaccording to claim 5, and further comprising a surfactant.
 18. A fabricsoftening composition containing one or more purified enzymes obtainedfrom the protein fraction according to claim
 5. 19. A composition forthe enzymatic treatment of cellulosic fibers or cellulosic fabrics,comprising a cellulase whose amino acid sequence is encoded by a nucleicacid sequence from a wild-type or mutant fungus of the genusChrysosporium, said composition having a pH between about 8.0 and about12.0, wherein the fungus is selected from the group consisting ofChrysosporium lucknowense, Chrysosporium pannorum, Chrysosporiumpruinosum, Chrysosporium keratinophilum, Chrysosporium lobatum,Chrysosporium merdarium, Chrysosporium queenslandicum, and Chrysosporiumtropicum.
 20. A composition according to claim 19, wherein the cellulaseis isolated or obtained from a wild-type or mutant fungus of the genusChrysosporium.